Treatment of antiviral gene related diseases by inhibition of natural antisense transcript to an antiviral gene

ABSTRACT

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of an Antiviral gene, in particular, by targeting natural antisense polynucleotides of an Antiviral gene. The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of Antiviral genes.

FIELD OF THE INVENTION

The present Application is a Divisional of U.S. Ser. No. 14/297,101filed Jun. 5, 2014, now U.S. Pat. No. 9,133,456, which is a Divisionalof application Ser. No. 13/321,996 filed Nov. 22, 2011, now U.S. Pat.No. 8,791,085, which is a 371 of International Application No.PCT/US2010/036558 filed May 28, 2010, which claims the priority to U.S.Provisional Patent Application Nos. 61/181,773 filed May 28, 2009,61/231,458 filed Aug. 5, 2009 and 61/237,886 filed Aug. 28, 2009, whichare all incorporated herein by reference in their entireties.

BACKGROUND

DNA-RNA and RNA-RNA hybridization are important to many aspects ofnucleic acid function including DNA replication, transcription, andtranslation. Hybridization is also central to a variety of technologiesthat either detect a particular nucleic acid or alter its expression.Antisense nucleotides, for example, disrupt gene expression byhybridizing to target RNA, thereby interfering with RNA splicing,transcription, translation, and replication. Antisense DNA has the addedfeature that DNA-RNA hybrids serve as a substrate for digestion byribonuclease H, an activity that is present in most cell types.Antisense molecules can be delivered into cells, as is the case foroligodcoxynucleotides (ODNs), or they can be expressed from endogenousgenes as RNA molecules. The FDA recently approved an antisense drug,VITRAVENE™ (for treatment of cytomegalovirus retinitis), reflecting thatantisense has therapeutic utility.

SUMMARY

In one embodiment, the invention provides methods for inhibiting theaction of a natural antisense transcript by using antisenseoligonucleotide(s) targeted to any region of the natural antisensetranscript resulting in up-regulation of the corresponding sense gene.It is also contemplated herein that inhibition of the natural antisensetranscript can be achieved by siRNA, ribozymes and small molecules,which are considered to be within the scope of the present invention.

One embodiment provides a method of modulating function and/orexpression of an Antiviral gene polynucleotide in patient cells ortissues in vivo or in vitro comprising contacting said cells or tissueswith an antisense oligonucleotide 5 to 30 nucleotides in length whereinsaid oligonucleotide has at least 50% sequence identity to a reversecomplement of a polynucleotide comprising 5 to 30 consecutivenucleotides within nucleotides 1 to 1267 of SEQ ID SEQ ID NO: 4, 1 to586 of SEQ ID NO: 5, 1 to 741 of SEQ ID SEQ ID NO: 6, 1 to 251 of SEQ IDNO: 7, 1 to 681 of SEQ ID NO: 8, and 1 to 580 of SEQ ID NO: 9 therebymodulating function and/or expression of the Antiviral genepolynucleotide in patient cells or tissues in vivo or in vitro.

In another embodiment, an oligonucleotide targets a natural antisensesequence of an Antiviral gene polynucleotide, for example, nucleotidesset forth in SEQ ID NO: 4 to 9, and any variants, alleles, homologs,mutants, derivatives, fragments and complementary sequences thereto.Examples of antisense oligonucleotides are set forth as SEQ ID NOS: 10to 30.

Another embodiment provides a method of modulating function and/orexpression of an Antiviral gene polynucleotide in patient cells ortissues in vivo or in vitro comprising contacting said cells or tissueswith an antisense oligonucleotide 5 to 30 nucleotides in length whereinsaid oligonucleotide has at least 50% sequence identity to a reversecomplement of the an antisense of the Antiviral gene polynucleotide;thereby modulating function and/or expression of the Antiviral genepolynucleotide in patient cells or tissues in vivo or in vitro.

Another embodiment provides a method of modulating function and/orexpression of an Antiviral gene polynucleotide in patient cells ortissues in vivo or in vitro comprising contacting said cells or tissueswith an antisense oligonucleotide 5 to 30 nucleotides in length whereinsaid oligonucleotide has at least 50% sequence identity to an antisenseoligonucleotide to an Antiviral gene antisense polynucleotide; therebymodulating function and/or expression of the Antiviral genepolynucleotide in patient cells or tissues in vivo or in vitro

In one embodiment, a composition comprises one or more antisenseoligonucleotides which bind to sense and/or antisense Antiviral genepolynucleotides.

In another embodiment, the oligonucleotides comprise one or moremodified or substituted nucleotides.

In another embodiment, the oligonucleotides comprise one or moremodified bonds.

In yet another embodiment, the modified nucleotides comprise modifiedbases comprising phosphorothioate, methylphosphonate, peptide nucleicacids, 2′-O-methyl, fluoro- or carbon, methylene or other locked nucleicacid (LNA) molecules. Preferably, the modified nucleotides are lockednucleic acid molecules, including α-L-LNA.

In another embodiment, the oligonucleotides are administered to apatient subcutaneously, intramuscularly, intravenously orintraperitoneally.

In another embodiment, the oligonucleotides are administered in apharmaceutical composition. A treatment regimen comprises administeringthe antisense compounds at least once to patient; however, thistreatment can be modified to include multiple doses over a period oftime. The treatment can be combined with one or more other types oftherapies.

In another embodiment, the oligonucleotides are encapsulated in aliposome or attached to a carrier molecule (e.g. cholesterol, TATpeptide).

Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of real time PCR results showing the foldchange+standard deviation in RIG1 mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real Time PCR results show that levels ofRIG1 mRNA in HepG2 cells are significantly increased 48 h aftertreatment with siRNAs to RIG1 antisense hs.601664 and AK300104. Barsdenoted as CUR-0544, CUR-0546, CUR-0548, CUR-0550, CUR-0552, CUR-0554,CUR-0556, CUR-0558 and CUR-0560 correspond to samples treated with SEQID NOS: 10 to 18 respectively.

FIG. 2 is a graph of real time PCR results showing the foldchange+standard deviation in MDA5 mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof MDA5 mRNA in HepG2 cells are significantly increased 48 h aftertreatment with two of the oligos designed to MDA5 antisense BU663736.Bars denoted as CUR-0908, CUR-0909, CUR-0910, CUR-0911, CUR-0912,CUR-0913, CUR-0914 and CUR-0915 correspond to samples treated with SEQID NOS: 19 to 26 respectively.

FIG. 3 is a graph of real time PCR results showing the foldchange+standard deviation in IFNA1 mRNA after treatment of HepG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof IFNA1 mRNA in HepG2 cells are significantly increased 48 h aftertreatment with one oligo designed to IFNA1 antisense DA393812. Barsdenoted as CUR-0916, CUR-0917, CUR-0918 and CUR-0919 correspond tosamples treated with SEQ ID NOS: 27 to 30 respectively.

FIG. 4 is a graph of real time PCR results showing the foldchange+standard deviation in IFNA1 mRNA after treatment of ZR75 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof IFNA1 mRNA in ZR75 cells are significantly increased 48 h aftertreatment with one oligo designed to IFNA1 antisense DA393812. Barsdenoted as CUR-0916, CUR-0919, CUR-0918 and CUR-0917 and correspond tosamples treated with SEQ ID NOS: 27, 30, 29 and 28 respectively.

FIG. 5 is a graph of real time PCR results showing the foldchange+standard deviation in IFNA1 mRNA after treatment of HUVEC cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof IFNA1 mRNA in HUVEC cells are significantly increased 48 h aftertreatment with one oligo designed to IFNA1 antisense DA393812. Barsdenoted as CUR-0916, CUR-0917, CUR-0918 and CUR-0919 correspond tosamples treated with SEQ ID NOS: 27 to 30 respectively.

FIG. 6: ELISA Assay results show that IFNA protein was upregulated inextracts from MCF7 cells which were treated with oligos designed toIFNA1 antisense DA393812. Bars denoted as CUR-0916, CUR-0919, CUR-0918and CUR-0917 and correspond to samples treated with SEQ ID NOS: 27, 30,29 and 28 respectively.

SEQUENCE LISTING DESCRIPTION

-   SEQ ID NO: 1: Homo sapiens (RIG1) mRNA (NCBI Accession No.    NM_014314.3).-   SEQ ID NO: 2: Homo sapiens interferon induced with helicase C domain    1 (IFIH1), mRNA (NCBI Accession No.: NM_022168.2).-   SEQ ID NO: 3: NM_024013.1 Homo sapiens interferon, alpha 1 ((IFNA1),    mRNA (NCBI Accession No.: NM_005172).-   SEQ ID NOS: 4-9: SEQ ID NO: 4: Natural RIG1 antisense sequence    (AK300104); SEQ ID NO: 5: Natural RIG1 antisense sequence    (hs.601664); SEQ ID NO; 6: Natural RIG1 antisense sequence    (hs.104091); SEQ ID NO: 7: Natural MMDA5 antisense sequence    (Hs.692345); SEQ ID NO: 8: Natural MDA5 antisense sequence    (BU663736) and SEQ ID NO: 9: natural IFNA1 antisense sequence    (DA393812)-   SEQ ID NOS: 10-30: antisense oligonucleotides. ‘r’ indicates RNA    and * indicates phosphorthioate bond.-   SEQ ID NOS: 31 and 39 are the reverse complements of the antisense    oligonucleotides SEQ ID NOS: 10 and 18 respectively. ‘r’ indicates    RNA.

DETAILED DESCRIPTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the ordering ofacts or events, as some acts may occur in different orders and/orconcurrently with other acts or events. Furthermore, not all illustratedacts or events are required to implement a methodology in accordancewith the present invention.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the genes disclosedherein, which in some embodiments relate to mammalian nucleic acid andamino acid sequences are intended to encompass homologous and/ororthologous genes and gene products from other animals including, butnot limited to other mammals, fish, amphibians, reptiles, and birds. Inembodiments, the genes or nucleic acid sequences are human.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a,”, “an” and “the” ore intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As used herein, the term “mRNA” means the presently known mRNAtranscript(s) of a targeted gene, and any further transcripts which maybe elucidated.

By “antisense oligonucleotides” or “antisense compound” is meant an RNAor DNA molecule that binds to another RNA or DNA (target RNA, DNA). Forexample, if it is an RNA oligonucleotide it binds to another RNA targetby means of RNA-RNA interactions and alters the activity of the targetRNA, An antisense oligonucleotide can upregulate or downregulateexpression and/or function of a particular polynucleotide. Thedefinition is meant to include any foreign RNA or DNA molecule which isuseful from a therapeutic, diagnostic, or other viewpoint. Suchmolecules include, for example, antisense RNA or DNA molecules,interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA,enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA,antisense oligomeric compounds, antisense oligonucleotides, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds that hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, partiallysingle-stranded, or circular oligomeric compounds.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. The term “oligonucleotide”, alsoincludes linear or circular oligomers of natural and/or modifiedmonomers or linkages, including deoxyribonucelcosides, ribonucleosides,substituted and alpha-anomeric forms thereof, peptide nucleic acids(PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate,and the like. Oligonucleotides are capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing. Hoögsteen orreverse Hoögsteen types of base pairing, or the like.

The oligonucleotide may be “chimeric”, that is, composed of differentregions. In the context of this invention “chimeric” compounds areoligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one monomer unit i.e., a nucleotide in thecase of an oligonucleotides compound. These oligonucleotides typicallycomprise at least one region wherein the oligonucleotide is modified inorder to exhibit one or more desired properties. The desired propertiesof the oligonucleotide include, but are not limited, for example, toincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Differentregions of the oligonucleotide may therefore have different properties.The chimeric oligonucleotides of the present invention can be formed asmixed structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide analogs asdescribed above.

The oligonucleotide can be composed of regions that can be linked in“register” that is, when the monomers are linked consecutively, as innative DNA, or linked via spacers. The spacers are intended toconstitute a covalent “bridge” between the regions and have in cases alength not exceeding about 100 carbon atoms. The spacers may carrydifferent functionalities, for example, having positive or negativecharge, carry special nucleic acid binding properties (intercalators,groove binders, toxins, fluorophors etc.), being lipophilic, inducingspecial secondary structures like, for example, alanine containingpeptides that induce alpha-helices.

As used herein “Antiviral genes” and “Antiviral gene” are inclusive ofall family members, mutants, alleles, fragments, species, coding andnoncoding sequences, sense and antisense polynucleotide strands, etc.

As used herein, the words RIG1, RIG-1, RIG-I, DEAD box protein 58,DKFZp434J1111, DKFZp68N19181, FLJ13599. Retinoic acid-inducible gene 1protein, Retinoic acid-inducible gene 1 protein, are considered the samein the literature and are used interchangeably in the presentapplication.

As used herein, the words MDA5, MDA-5, Melanomadifferentation-associated protein 5, Helicard, Helicase with 2 CARDdomains. Hlcd. IDDM19, Interferon-induced helicase C domain-containingprotein 1, Interferon-induced with helicase C domain protein 1,MGC133047, Murabutide down-regulated protein, RH116, RNA helicase-DEADbox protein 116 are considered the same in the literature and are usedinterchangeably in the present application.

As used herein, the words IFNA1, IFNAI1. G10P1, GARG-16, IFI56, IFI-56,IFI-56K, IFIT-1; Interferon-induced 56 kDa protein, Interferon-inducedprotein with tetratricopeptide repeats 1, ISG56, RNM561 are consideredthe same in the literature and are used interchangeably in the presentapplication.

As used herein, the term “oligonucleotide specific for” or“oligonucleotide which targets” refers to an oligonucleotide having asequence (i) capable of forming a stable complex with a portion of thetargeted gene, or (ii) capable of forming a stable duplex with a portionof a mRNA transcript of the targeted gene. Stability of the complexesand duplexes can be determined by theoretical calculations and/or invitro assays. Exemplary assays for determining stability ofhybridization complexes and duplexes are described in the Examplesbelow.

As used herein, the term “target nucleic acid” encompasses DNA, RNA(comprising premRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA, coding, noncoding sequences, sense or antisensepolynucleotides. The specific hybridization of an oligomeric compoundwith its target nucleic acid interferes with the normal function of thenucleic acid. This modulation of function of a target nucleic acid bycompounds, which specifically hybridize to it, is generally referred toas “antisense”. The functions of DNA to be interfered include, forexample, replication and transcription. The functions of RNA to beinterfered, include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in or facilitatedby the RNA. The overall effect of such interference with target nucleicacid function is modulation of the expression of an encoded product oroligonucleotides.

RNA interference “RNAi” is mediated by double stranded RNA (dsRNA)molecules that have sequence-specific homology to their “target” nucleicacid sequences. In certain embodiments of the present invention, themediators are 5-25 nucleotide “small interfering” RNA duplexes (siRNAs).The siRNAs are derived from the processing of dsRNA by an RNase enzymeknown as Dicer. siRNA duplex products are recruited into a multi-proteinsiRNA complex termed RISC (RNA Induced Silencing Complex). Withoutwishing to be bound by any particular theory, a RISC is then believed tobe guided to a target nucleic acid (suitably mRNA), where the siRNAduplex interacts in a sequence-specific way to mediate cleavage in acatalytic fashion. Small interfering RNAs that can be used in accordancewith the present invention can be synthesized and used according toprocedures that are well known in the art and that will be familiar tothe ordinarily skilled artisan. Small interfering RNAs for use in themethods of the present invention suitably comprise between about 1 toabout 50 nucleotides (nt). In examples of non limiting embodiments.siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt,about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25nucleotides.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the ease of genes that have not been sequenced.Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

By “enzymatic RNA” is meant an RNA molecule with enzymatic activity.Enzymatic nucleic acids (ribozymes) act by first binding to a targetRNA. Such binding occurs through the target binding portion of anenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid fuel recognizes and then binds a target RNAthrough base pairing, and once bound to the correct site, actsenzymatically to cut the target RNA.

By “decoy RNA” is meant an RNA molecule that mimics the natural bindingdomain for a ligand. The decoy RNA therefore competes with naturalbinding target for the binding of a specific ligand. For example, it hasbeen shown that over-expression of HIV trans-activation response (TAR)RNA can act as a “decoy” and efficiently binds HIV tat protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA. Thisis meant to be a specific example. Those in the art will recognize thatthis is but one example, and other embodiments can be readily generatedusing techniques generally known in the art.

As used herein, the term “monomers” typically indicates monomers linkedby phosphodiester bonds or analogs thereof to form oligonucleotidesranging in size from a few monomeric units, e.g., from about 3-4, toabout several hundreds of monomeric units. Analogs of phosphodiesterlinkages include: phosphorothioate, phosphorodithioate,methylphosphomates, phosphoroselenoate, phosphoramidate, and the like,as more fully described below.

The term “nucleotide” covers naturally occurring nucleotides as well asnonnaturally occurring nucleotides. It should be clear to the personskilled in the art that various nucleotides which previously have beenconsidered “non-naturally occurring” have subsequently been found innature. Thus, “nucleotides” includes not only the known purine andpyrimidine heterocycles-containing molecules, but also heterocyclicanalogues and tautomers thereof. Illustrative examples of other types ofnucleotides are molecules containing adenine, guanine, thymine,cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin,N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine and the “non-naturally occurring” nucleotidesdescribed in U.S. Pat No. 5,432,272. The term “nucleotide” is intendedto cover every and all of these examples as well as analogues andtautomers thereof. Especially interesting nucleotides ate thosecontaining adenine, guanine, thymine, cytosine, and uracil, which areconsidered as the naturally occurring nucleotides in relation totherapeutic and diagnostic application in humans. Nucleotides includethe natural 2′-deoxy and 2′-hydroxyl sugars, e.g., as described inKornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco,1992) as well as their analogs.

“Analogs” in reference to nucleotides includes synthetic nucleotidesleaving modified base moieties and/or modified sugar moieties. Suchanalogs include synthetic nucleotides designed to enhance bindingproperties, e.g., duplex or triplex stability, specificity, or the like.

As used herein, “hybridization” means the pairing of substantiallycomplementary strands of oligomeric compounds. One mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoögsteen orreversed Hoögsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleotides) of the strands of oligomeric compounds.For example, adenine and thymine are complementary nucleotides whichpair through the formation of hydrogen bonds. Hybridization can occurunder varying circumstances.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated. In general, stringent hybridization conditionscomprise low concentrations (<0.15 M) of salts with inorganic cationssuch as Na++ or K++ (i.e., low ionic strength), temperature higher than20° C.-25° C. below the Tm of the oligomeric compound:target sequencecomplex, and the presence of denaturants such as formamide,dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecylsulfate (SDS). For example, the hybridization rate decreases 1.1% foreach 1% formamide. An example of a high stringency hybridizationcondition is 0.1× sodium chloride-sodium citrate buffer(SSC)/0.1% (w/v)SDS at 60° C. for 30 minutes.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides on one or two oligomeric strands. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, said target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleotides such that stable and specificbinding occurs between the oligomeric compound and a target nucleicacid.

It is understood in the art that the sequence of tin oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligomeric compounds of the presentinvention comprise at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an antisense compound in which 18 of 20nucleotides of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remaining noncomplementarynucleotides may be clustered or interspersed with complementarynucleotides and need not be contiguous to each other or to complementarynucleotides. As such, an antisense compound which is 18 nucleotides inlength having 4 (four) noncomplementary nucleotides which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art.Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., (1981) 2, 482-489).

As used herein, the term “Thermal Melting Point (Tm)” refers to thetemperature, under defined ionic strength, pH, and nucleic acidconcentration, at which 50% of the oligonucleotides complementary to thetarget sequence hybridize to the target sequence at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is at least about 0.01 to 1.0 M Na ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

As used herein, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene.

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype gene products. Variants may result from at least one mutation inthe nucleic acid sequence and may result in altered mRNAs or inpolypeptides whose structure or function may or may not be altered Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility versus resistance.

Derivative polynucleotides include nucleic acids subjected to chemicalmodification, for example, replacement of hydrogen by an alkyl, acyl, oramino group. Derivatives, e.g., derivative oligonucleotides, maycomprise non-naturally-occurring portions, such as altered sugarmoieties or inter-sugar linkages. Exemplary among these arephosphorothioate and other sulfur containing species which are known inthe art. Derivative nucleic acids may also contain labels, includingradionucleotides, enzymes, fluorescent agents, chemiluminescent agents,chromogenic agents, substrates, cofactors, inhibitors, magneticparticles, and the like.

A “derivative” polypeptide or peptide is one that is modified, forexample, by glycosylation, pegylation, phosphorylation, sulfation,reduction/alkylation, acylation, chemical coupling, or mild formalintreatment. A derivative may also be modified to contain a detectablelabel, either directly or indirectly, including, but not limited to, aradioisotope, fluorescent, and enzyme label.

As used herein, the term “animal” or “patient” is meant to include, forexample, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys,horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken,reptiles, fish, insects and arachnids.

“Mammal” covers warm blooded mammals that are typically under medicalcare (e.g., humans and domesticated animals). Examples include feline,canine, equine, bovine, and human, as well as just human.

“Treating” or “treatment” covers the treatment of a disease-state in amammal, and includes: (a) preventing the disease-state from occurring ina mammal, in particular, when such mammal is predisposed to thedisease-state but has not yet been diagnosed as having it; (b)inhibiting the disease-state, e.g., arresting it development: and/or (c)relieving the disease-state, e.g., causing regression of the diseasestate until a desired endpoint is reached. Treating also includes theamelioration of a symptom of a disease (e.g., lessen the pain ordiscomfort), wherein such amelioration may or may not be directlyaffecting the disease (e.g., cause, transmission, expression, etc.).

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in mammals, including, but not limited to:leukemias, lymphomas, melanomas, carcinomas and sarcomas. The cancermanifests itself as a “tumor” or tissue comprising malignant cells ofthe cancer. Examples of tumors include sarcomas and carcinomas such as,but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's rumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Additional cancers which can be treated by the disclosedcomposition according to the invention include but not limited to, forexample, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,adrenal cortical cancer, and prostate cancer.

“Neurological disease or disorder” refers to any disease or disorder ofthe nervous system and/or visual system. “Neurological disease ordisorder” include disease or disorders that involve the central nervoussystem (brain, brainstem and cerebellum), the peripheral nervous system(including cranial nerves), and the autonomic nervous system (parts ofwhich are located in both central and peripheral nervous system).Examples of neurological disorders include but are not limited to,headache, stupor and coma, dementia, seizure, sleep disorders, trauma,infections, neoplasms, neuroopthalmology, movement disorders,demyelinating diseases, spinal cord disorders, and disorders ofperipheral nerves, muscle and neuromuscular junctions. Addiction andmental illness, include, but are not limited to, bipolar disorder andschizophrenia, are also included in the definition of neurologicaldisorder. The following is a list of several neurological disorders,symptoms, signs and syndromes that can be treated using compositions andmethods according to the present invention: acquired epileptiformaphasia; acute disseminated encephalomyclitis; adrenoleukodystrophy;age-related macular degeneration; agenesis of the corpus callosum;agnosia; Aicardi syndrome; Alexander disease; Alpers' disease;alternating hemiplegia; Vascular dementia; amyotrophic lateralsclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia;aphasia; apraxia; arachnoid cysts; arachnoiditis; Anroni-Chiarimalformation; arteriovenous malformation; Asperger syndrome; ataxiatelegiectasia; attention deficit hyperactivity disorder; autism;autonomic dysfunction; back pain; Batten disease; Behcet's disease;Bell's palsy; benign essential blepharospasm; benign focal; amyotrophy;benign intracranial hypertension; Binswanger's disease; blepharospasm;Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; braininjury; brain tumors (including glioblastoma multiforme); spinal tumor;Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome;causalgia; central pain syndrome; central pontine myelinolysis: cephalicdisorder; cerebral aneurysm; cerebral arteriosclerosis; cerebralatrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Toothdisease; chemotherapy-induced neuropathy and neuropathic pain; Chiarimalformation; chorea; chronic inflammatory demyclinating polyneuropathy:chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome;coma, including persistent vegetative state; congenital facial diplegia;corticobasal degeneration; cranial arteritis; craniosynostosis;Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing'ssyndrome; cytomegalic inclusion body disease; cytomegalovirus infection;dancing eyes-dancing feet syndrome; DandyWalker syndrome; Dawsondisease; De Morsier's syndrome; Dejerine-Klumke palsy; dementia;dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia;dysgraphia; dyslexia; dystonias; early infantile epilepticencephalopathy; empty sella syndrome; encephalitis; encephaloceles;encephalotrigeminal angiomatosis; epilepsy, Erb's palsy; essentialtremor, Fabry's disease; Fahr's syndrome; fainting; familial spasticparalysis; febrile seizures; Fisher syndrome; Friedreich's ataxia;fromo-temporal dementia and other “tauopathies”; Gaucher's disease;Gerstmann's syndrome; giant cell arteritis; giant cell inclusiondisease; globoid cell leukodystrophy, Guillain-Barre syndrome;HTLV-1-associated myelopathy; Hallervorden-Spatz disease; head injury;headache; hemifacial spasm; hereditary spastic paraplegia; heredopathiaatactic a polyneuritiformis; herpes zoster oticus; herpes zoster;Hirayama syndrome; HIV associated dementia and neuropathy (alsoneurological manifestations of AIDS); holoprosencephaly; Huntington'sdisease and other polyglutamine repeat diseases; hydranencephaly;hydrocephalus; hypercorrisolism; hypoxia, immune-mediatedencephalomyelitis; inclusion body myositis; incontinentia pigmenti;infantile phytanic acid storage disease; infantile refsum disease;infantile spasms; inflammatory myopathy; intracranial cyst; intracranialhypertension; Joubert syndrome; Keams-Sayre syndrome; Kennedy diseaseKinsbourne syndrome; Klippel Feil syndrome; Krabbe disease;Kugelberg-Welander disease; kuru; Lafora disease; Lambert-Eatonmyasthenic syndrome; Landau-Kleffner syndrome; lateral medullary(Wallenberg) syndrome; learning disabilities; Leigh's disease;Lennox-Gustaut syndrome; Lesch-Nyhan syndrome: leukodystrophy; Lewy bodydementia; Lissencephaly; locked-in syndrome, Lou Gehrig's disease (i.e.,motor neuron disease or amyotrophic lateral sclerosis); lumbar discdisease; Lyme disease-neurological sequelae; Machado-Joseph disease;macrencephaly; megalenscephaly; Melkersson-Rosenthal syndrome; Menieresdisease; meningitis; Menkes disease; metachromatic leukodystrophy;microcephaly; migraine; Miller Fisher syndrome; mini-strokes;mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motorneuron disease; Moyamoya disease; mucopolysaccharidoses; milti-infaretdementia; multifocal motor neuropathy; multiple sclerosis and otherdemyelinating disorders: multiple system atrophy with posturalhypotension; p muscular dystrophy; myasthenia gravis; myelinoclasticdiffuse sclerosis; myoclonic encephalopathy of infants; myoclonus:myopathy; myotonia congenital; narcolepsy; neurofibromatosis;neuroleptic malignant syndrome: neurological manifestations of AIDS;neurological sequelae oflupus; neuromyotonia; neuronal ceroidlipofuscinosis; neuronal migration disorders; Niemann-Pick disease;O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinaldysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy;opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overusesyndrome; paresthesia; Neurodegenerative disease or disorder(Parkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), dementia, multiple sclerosis andother diseases and disorders associated with neuronal cell death);paramyotonia congenital; paraneoplastic diseases; paroxysmal attacks;Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodicparalyses; peripheral neuropathy; painful neuropathy and neuropathicpain; persistent vegetative state; pervasive developmental disorders;photic sneeze reflex, phytanic acid storage disease: Pick's disease;pinched nerve; pituitary tumors; polymyositis; porencephaly: post-poliosyndrome; postherpetic neuralgia; postinfectious encephalomyelitis;postural hypotension; Prader-Willi syndrome; primary lateral sclerosis;prion diseases; progressive hemifacial atrophy; progressivemultifocalleukoencephalopathy; progressive sclerosing poliodystrophy;progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Huntsyndrome (types I and II); Rasmussen's encephalitis; reflex sympatheticdystrophy syndrome; Refsum disease; repetitive motion disorders;repetitive stress injuries; restless legs syndrome;retrovirus-associated myclopathy; Rett syndrome; Reye's syndrome; SaintVitus dance; Sandhoff disease; Schilders disease; schizencephaly;septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Dragersyndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome; spasticity;spina bifida; spinal cord injury; spinal cord tumors; spinal muscularatrophy; Stiff-Person syndrome; stroke; Sturge-Weber syndrome; subacutesclerosing panencephalitis: subcortical arteriosclerotic encephalopathy;Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachsdisease; temporal arteritis; tethered spinal cord syndrome; Thomsondisease; thoracic outlet syndrome; Tic Douloureux; Todd's paralysis;Tourette syndrome; transient ischemic attack; transmissible spongiformencephalopathies; transverse myelitis; traumatic brain injury; tremor;trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis;vascular dementia (multi-infarct dementia); vasculitis includingtemporal arteritis; Von Hippel-Lindau disease; Wallenberg's syndrome;Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome;Wildon's disease; and Zellweger syndrome.

An “Inflammation” refers to systemic inflammatory conditions andconditions associated locally with migration and attraction ofmonocytes, leukocytes and/or neutrophils. Examples of inflammationinclude, but are not limited to. Inflammation resulting from infectionwith pathogenic organisms (including gram-positive bacteria,gram-negative bacteria, viruses, fungi, and parasites such as protozoaand helminths), transplant rejection (including rejection of solidorgans such as kidney, liver, heart, lung or cornea, as well asrejection of bone marrow transplants including graft-versus-host disease(GVHD)), or from localized chronic or acute autoimmune or allergicreactions. Autoimmune diseases include acute glomerulonephritis;rheumatoid or reactive arthritis; chronic glomerulonephritis;inflammatory bowel diseases such as Crohn's disease, ulcerative colitisand necrotizing enterocolitis; granulocyte transfusion associatedsyndromes: inflammatory dermatoses such as contact dermatitis, atopicdermatitis, psonasis; systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, and some forms of diabetes, or anyother autoimmune state where attack by the subject's own immune systemresults in pathologic tissue destruction. Allergic reactions includeallergic asthma, chronic bronchitis, acute and delayed hypersensitivity.Systemic inflammatory disease states include inflammation associatedwith trauma, burns, reperfusion following ischemic events (e.g.thrombotic events in heart, brain, intestines or peripheral vasculature,including myocardial infarction and stroke), sepsis, ARDS or multipleorgan dysfunction syndrome. Inflammatory cell recruitment also occurs inatherosclerotic plaques. Inflammation includes, but is not limited to,Non-Hodgkin's lymphoma, Wegener's granulomatosis, Hashimoto'sthyroiditis, hepatocellular carcinoma, thymus atrophy, chronicpancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia,osteoarthritis, ulcerative colitis, papillary carcinoma, Crohn'sdisease, ulcerative colitis, acute cholecystitis, chronic cholecystitis,cirrhosis, chronic sialadenitis, peritonitis, acute pancreatitis,chronic pancreatitis, chronic Gastritis, adenomyosis, endometriosis,acute cervicitis, chronic cervicitis, lymphoid hyperplasia, multiplesclerosis, hypertrophy secondary to idiopathic thrombocytopenic purpura,primary IgA nephropathy, systemic lupus erythematosus, psoriasis,pulmonary emphysema, chronic pyclonephritis, and chronic cystitis.

A cardiovascular disease or disorder includes those disorders that caneither cause ischemia or are caused by reperfusion of the heart.Examples include, but are not limited to, atherosclerosis, coronaryartery disease, granulomatous myocarditis, chronic myocarditis(non-granulotmatous), primary hypertrophic cardiomyopathy, peripheralartery disease (PAD), stroke, angina pectoris, myocardial infarction,cardiovascular tissue damage caused by cardiac arrest, cardiovasculartissue damage caused by cardiac bypass, cardiogenic shock, and relatedconditions that would be known by those of ordinary skill in the art orwhich involve dysfunction of or tissue damage to the heart orvasculature, especially, but not limited to, tissue damage related to anAntiviral gene activation. CVS diseases include, but are not limited to,atherosclerosis, granulomatous myocarditis, myocardial infarction,myocardial fibrosis secondary to valvular heart disease, myocardialfibrosis without infarction, primary hypertrophic cardiomyopathy, andchronic myocarditis (non-granulomatous).

Examples of viruses or viruses causing patient diseases comprise:Herpesviruses, such as for example, Alpha-herpesviruses (e.g. Herpessimplex virus 1 (HSV-1), Herpes simplex virus 2 (HSV-2): VaricellaZoster virus (VZV)), Beta-herpesviruses (e.g. Cytomegalovirus (CMV);Herpes virus 6 (HHV-6)), Gamma-herpesviruses (e.g. Epstein-Barr virus(EBV); Herpes virus 8 (HHV-8)), Hepatitis viruses (e.g. Hepatitis Avirus; Hepatitis 8 virus; Hepatitis C virus; Hepatitis D virus;Hepatitis E virus). Retroviruses (e.g. Human Immunodeficiency 1(HIV-1)), Orthomyxoviruses (e.g. Influenza viruses A, B and C),Paramyxoviruses, Respiratory Syncytial virus (RSV), Parainfluenzaviruses (PI) (e.g. Mumps virus, Measles virus), Togaviruses (e.g.Rubella virus), Picomaviruses (e.g. Enteroviruses, Rhinoviruses;Coronavirses), Papovaviruses (e.g. Human papilloma viruses (HPV):Polyomaviruses (BKV and ICV); Gastroenteritis viruses), Filoviridae,Bunyaviridae, Rhabdoviridae, Flaviviridae.

Examples of bacteria causing bacterial diseases comprise: Staphylococcusaureus; strains include methicillin resistant (MRSA),methicillin-vancomycin resistant (VMRSA) and vancomycin intermediateresistant (VISA); Staphylococcus epidermidis; Enterococcus faecalis andE. faccium: strains include vancomycin resistant (VRE); Streptococcuspneumoniae; Pseudomonas aeruginosa; Burkholdia cepacia; Xanthomonasmaltophila; Escherichia coli; Enterobacter spp.; Klebsiella pneumoniae;Salmonella spp.

Polynucleotide and Oligonucleotide Compositions and Molecules

Targets

In one embodiment, the targets comprise nucleic acid sequences of anAntiviral gene, including without limitation sense and/or antisensenoncoding and/or coding sequences associated with an Antiviral gene.

In one embodiment, the targets comprise nucleic acid sequences of RIG1,including without limitation sense and/or antisense noncoding and/orcoding sequences associated with RIG1 gene.

In one embodiment, the targets comprise nucleic acid sequences of MDA5,including without limitation sense and/or antisense noncoding and/orcoding sequences associated with MDA5 gene.

In one embodiment, the targets comprise nucleic acid sequences of IFNA1,including without limitation sense and/or antisense noncoding and/orcoding sequences associated with IFNA1 gene.

The retinoic acid-inducible gene 1 product (RIG-I) has been identifiedas a cellular sensor of RNA virus infection resulting in beta interferon(IFN-B) induction. However, many viruses are known to encode viralproducts that inhibit IFN-B production. In the case of influenza Avirus, the viral nonstructural protein 1 (NS 1) prevents the inductionof the IFN-B promoter by inhibiting the activation of transcriptionfactors, including IRF-3, involved in IFN-B transcriptional activation.The inhibitory properties of NS1 appear to be due at least in part toits binding to double-stranded RNA (dsRNA), resulting in thesequestration of this viral mediator of RIG-I activation.

Retinoic acid-inducible gene 1 (RIG-I) and melanomadifferentiation-associated gene 5 (MDA5) are cytoplasmic DEx(D/H) boxhelicases that can detect intracellular viral products such as genomicRNA (vRNA) to signal IFN−/− production in infected cells. Signaling byeach occurs through homotypic caspase activation and recruitment domain(CARD)-interactions with the interferon promoter-stimulating factor 1(IPS-I) adaptor protein, which recruits RIG-I and MDA5 to the outermembrane of the mitochondria as part of a macromolecular signalingcomplex that serves to activate downstream interferon-regulatory factors(IRFs) and other transcription factors that induce IFN−/− and ISGexpression. Although RIG-I and MDA5 may share similar signaling featuresand structural homology, accumulating evidence suggests that the twohelicases may discriminate among different ligands to trigger the innateimmune response to RNA viruses. Signaling by RIG-I is triggered duringinfection by a number of RNA viruses and by the presence of syntheticRNA transcribed in vitro. More recently, RIG-I has been implicated inthe recognition of RNA moieties that harbor 5′ triphosphate ends or ofRNAs that assume complex secondary structures. In contrast, signaling byMDA5 is uniquely triggered during pieornavirus infections or in thepresence of a synthetic RNA polymer consisting of annealing strands ofinosine and cytosine, poly(1:C).

Human melanoma cells can be reprogrammed to terminally differentiate andirreversibly lose proliferative capacity by appropriate pharmacologicalmanipulation. Subtraction hybridization identified melanomadifferentiation-associated gene-5 (mda-5) as a gene induced duringdifferentiation, cancer reversion, and programmed cell death(apoptosis). This gene contains both a caspase recruitment domain andputative DExH group RNA helicase domains. Atypical helicase motifs ofMDA-5 deviate from consensus sequences but are well conserved in apotentially new group of cloned and hypothetical proteins, mda-5 is anearly response gene inducible by IFN and tumor necrosis factor-a,responding predominantly to IFN−. Protein kinase C activation bymezerein further augments mda-5 expression induced by IFN−. Expressionof mda-5 is controlled transcriptionally by IFN−. and the MDA-5 proteinlocalizes in the cytoplasm, mda-5 displays RNA-dependent ATPaseactivity, and ectopic expression of mda5 in human melanoma cellsinhibits colony formation. In these contexts, mda-5 may function as amediator of IFN-induced growth inhibition and/or apoptosis. MDA-5 is adouble-stranded RNA-dependent ATPase that contains both a caspaserecruitment domain and RNA helicase motifs, with a confirmed associationwith growth and differentiation in human melanoma cells.

Progress has been made in understanding the molecular basis of theantiviral actions of interferons (IFNs), as well as strategies evolvedby viruses to antagonize the actions of IFNs. Furthermore, advances madewhile elucidating the IFN system have contributed significantly to ourunderstanding in multiple areas of virology and molecular cell biology,ranging from pathways of signal transduction to the biochemicalmechanisms of transcriptional and translational control to the molecularbasis of viral pathogenesis. IFNs are approved therapeutics and havemoved from the basic research laboratory to the clinic. Among theIFN-induced proteins important in the antiviral actions of IFNs are theRNA-depersdent protein kinase (PKR), the 2′,5′-oligoadenylate synthetase(OAS) and RNase L, and the Mx protein GTPases. Double stranded RNA playsa central role in modulating protein phosphorylation and RNA degradationcatalyzed by the IFN-inducible PKR kinase and the2′-5′-oligoadenylate-dependent RNase L, respectively, and also in RNAediting by the IFN-inducible RNA-specific adenosine deaminase (ADARI).IFN also induces a form of inducible nitric oxide synthase (iNOS2) andthe major histocompatibility complex class I and II proteins, all ofwhich play important roles in immune response to infections. Severaladditional genes whose expression profiles are altered in response toIFN treatment and virus infection have been identified by microarrayanalyses. The availability of cDNA and genomic clones for many of thecomponents of the IFN system, including IFN-alpha, IFN-beta, andIFN-gamma, their receptors, Jak and Stat and IRF signal transductioncomponents, and proteins such as PKR, 2′,5′-OAS, Mx, and ADAR, whoseexpression, is regulated by IFNs, has permitted the generation of mutantproteins, cells that overexpress different forms of the proteins, andanimals in which their expression has been disrupted by targeted genedisruption. The use of these IFN system reagents, both in cell cultureand in whole animals, continues to provide important contributions toour understanding of the virus-host interaction and cellular antiviralresponse.

Interferons (IFN)s are involved in numerous immune interactions duringviral infections and contribute to both induction and regulation ofinnate and adaptive antiviral mechanisms. IFNs playa pivotal rule in theoutcome of a viral infection, as demonstrated by the impaired resistanceagainst different viruses in mice deficient for the receptors IFNAR-2and IFNGR. During viral infections, IFNs are involved to numerous immuneinteractions as inducers, regulators, and effectors of both innate andadaptive antiviral mechanisms IFN-alpha/beta is produced rapidly whenviral factors, such as envelope glycoproteins, CpG DNA, or dsRNA,interact with cellular pattern-recognition receptors (PRRs), such asmannose receptors, toll-like receptors (TLRs), and cytosolic receptors.These host-virus interactions signal downstream to activatetranscription factors needed to achieve expression from IFN-alpha/betagenes. These include IFN regulatory factor-3 (IRF-3), IRF-5, IRF-7,c-Jun/ATF-2. and NF-kappaB. In

contrast. IFN-gamma is induced by receptor-mediated stimulation or inresponse to early produced cytokines, including interleukin-2 (IL-12),IL-18, and IFN-alpha/beta, or by stimulation through T cell receptors(TCRs)or natural killer (NK) cell receptors. IFNs signal throughtransmembrane receptors, activating mainly Jak-Stat pathways but alsoother signal transduction pathways. Cytokine and TCR-induced IFN-gammaexpression uses distinct signal transduction pathways involving suchtranscription factors as NFAT, Stats and NF-kappaB. This results ininduction and activation of numerous intrinsic antiviral factors, suchas RNA-activated protein kinase (PKR), the 2-5A system, Mx proteins, andseveral apoptotic pathways. In addition, IFNs modulate distinct aspectsof both innate and adaptive immunity. Thus, IFNalpha/beta and IFN-gammaaffect activities of macrophages, NK cells, dendritic cells (DC), and Tcells by enhancing antigen presentation, cell trafficking, and celldifferentiation and expression profiles, ultimately resulting inenhanced antiviral effector functions.

In some embodiments, antisense oligonucleotides are used to prevent ortreat diseases or disorders associated with Antiviral gene familymembers. Exemplary Antiviral gene mediated diseases and disorders whichcan be treated with cell/tissues regenerated from stem cells obtainedusing the antisense compounds comprise: cancer, an inflammatory disease,a disease, disorder or a condition caused by an infectious agent (e.g.,including, viral, bacterial, fungal, protozoan etc.), a viral diseaseinflammation, arthritis, psoriasis, a neurological disease or disorder,an immune system disease or disorder, an autoimmune disease or disorder,an immunodeficiency disease, an allergy, psoriasis, neurologicaldiseases, a renal disease or disorder, a cardiovascular disease ordisorder, a muscular disease or disorder, atherosclerosis, diabetes, ahepatic disease or disorder and a communicable disease.

In another embodiment, the antisense oligonucleotides modulate theexpression, in vivo amounts and/or function of an Antiviral gene inpatients suffering from or at risk of developing diseases or disordersassociated with Antiviral genes.

In another example, Human metapneumovirus (HMPV) is a recentlydiscovered pathogen that causes a significant proportion of respiratoryinfections in young infants, the elderly and immunocompromised patients.Modulation of RIG-I expression and/or function modulate viral infectionby various mechanisms, including for example, viral genomic sequencedestruction. RIG-I antisense oligonucleotides would thus be of greattherapeutic value in the development of anti-viral agents.

In one embodiment, modulation of retinoic acid inducible gene 1 (RIG-I)detects and destroys viral genomes. In another embodiment, modulation ofretinoic acid inducible gene 1 (RIG-I) prevents or treats against viralinfections in patients.

In one embodiment, the oligonucleotides are specific for polynucleotidesof an Antiviral gene, which includes, without limitation noncodingregions. The Antiviral gene targets comprise variants of an Antiviralgene; mutants of an Antiviral gene, including SNPs; noncoding sequencesof an Antiviral gene; alleles, fragments and the like. Preferably theoligonucleotide is an antisense RNA molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to an Antiviral gene polynucleotides alone butextends to any of the isoforms, receptors, homologs, non-coding regionsand the like of an Antiviral gene.

In another embodiment, an oligonucleotide targets a natural antisensesequence (natural antisense to the coding and non-coding regions) of anAntiviral gene targets, including, without limitation, variants,alleles, homologs, mutants, derivatives, fragments and complementarysequences thereto. Preferably the oligonucleotide is an antisense RNA orDNA molecule.

In another embodiment, the oligomeric compounds of the present inventionalso include variants in which a different base is present at one ormore of the nucleotide positions in the compound. For example, if thefirst nucleotide is an adenine, variants may be produced which containthymidine, guanosine, cytidine or other natural or unnatural nucleotidesat this position. This may be done at any of the positions of theantisense compound.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 50% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%, In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired. Such conditionsinclude, i.e., physiological conditions in the case of in vivo assays ortherapeutic treatment, and conditions in which assays are performed inthe case of in vitro assays.

An antisense compound, whether DNA, RNA, chimeric, substituted etc, isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarily to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed.

In another embodiment, targeting of an Antiviral gene including withoutlimitation, antisense sequences which are identified and expanded, usingfor example. PCR, hybridization etc., one or more of the sequences setforth as SEQ ID NO: 4 to 9, and the like, modulate the expression orfunction of an Antiviral gene. In one embodiment, expression or functionis up-regulated as compared to a control. In another embodiment,expression or function is down-regulated as compared to a control.

In another embodiment, oligonucleotides comprise nucleic acid sequencesset forth as SEQ ID NOS: 10 to 30 including antisense sequences whichare identified and expanded, using for example, PCR, hybridization etc.These oligonucleotides can comprise one or more modified nucleotides,shorter or longer fragments, modified bonds and the like. Examples ofmodified bonds or internuclcotide linkages comprise phosphorothioate,phosphorodithioate or the like. In another embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides, per se, is also known andneed not be described here.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

In embodiments of the present invention oligomeric antisense compounds,particularly oligonucleotides, bind to target nucleic acid molecules andmodulate the expression and/or function of molecules encoded by a targetgene. The functions of DNA to be interfered comprise, for example,replication and transcription. The functions of RNA to be interferedcomprise all vital functions such as, for example, translocation of theRNA to the site of protein translation, translation of protein from theRNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The functions may be up-regulated or inhibited depending on thefunctions desired.

The antisense compounds, include, antisense oligomeric compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, primers, probes, and otheroligomeric compounds that hybridize to at least a portion of the targetnucleic acid. As such, these compounds may be introduced in the form ofsingle-stranded, double-stranded, partially single-stranded, or circularoligomeric compounds.

Targeting an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes an Antiviral gene.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

In one embodiment, the antisense oligonucleotides bind to the naturalantisense sequences of an Antiviral gene and modulate the expressionand/or function of an Antiviral gene (SEQ ID NO: 1 to 3). Examples ofantisense sequences include SEQ ID NOS: 4 to 30.

In another embodiment, the antisense oligonucleotides bind to one ormore segments of an Antiviral gene polynucleotide and modulate theexpression and/or function of an Antiviral gene. The segments compriseare least five consecutive nucleotides of an Antiviral gene sense orantisense polynucleotides.

In another embodiment, the antisense oligonucleotides are specific fornatural antisense sequences of an Antiviral gene wherein binding of theoligonucleotides to the natural antisense sequences of an Antiviral genemodulate expression and/or function of an Antiviral gene.

In another embodiment, oligonucleotide compounds comprise sequences setforth as SEQ ID NOS: 10 to 30, antisense sequences which are identifiedand expanded, using for example. PCR, hybridization etc. Theseoligonucleotides can comprise one or more modified nucleotides, shorteror longer fragments, modified bonds and the like. Examples of modifiedbonds or internucleotide linkages comprise phosphorothioate,phosphorodithioate or the like. In another embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides, per se, is also known andneed not be described here.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes has a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG; and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA transcribed from a geneencoding an Antiviral gene, regardless of the sequence(s) of suchcodons. A translation termination codon (or “stop codon”) of a gene mayhave one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions that may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, atargeted region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Another target region includes the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene). Still another target regionincludes the 3′ untranslated region (3′UTR), known in the art to referso the portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA (or correspondingnucleotides on the gene). The 5′ cap site of an mRNA comprises anN7-methylated guanosine residue joined to the 5-most residue of the mRNAvia a 5-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap site. Another target region for thisinvention is the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. In one embodiment, targeting splicesites, i.e., intron-exon junctions or exon-intron junctions, isparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. An aberrant fusion junction due to rearrangementor deletion is another embodiment of a target site. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. Introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

In another embodiment, the antisense oligonucleotides bind so codingand/or non-coding regions of a target polynucleotide and modulate theexpression and/or function of the target molecule.

In another embodiment, the antisense oligonucleotides bind to naturalantisense polynucleotides and modulate the expression and/or function ofthe target molecule.

In another embodiment, the antisense oligonucleotides bind to sensepolynucleotides and modulate the expression and/or function of thetarget molecule.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame genomic DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also embodiments of target nucleic acids.

The locations on the target nucleic acid to which the antisensecompounds hybridize are defined as at least a 5-nucleotide long portionof a target region to which an active antisense compound is targeted.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure.

Target segments 5-100 nucleotides in length comprising a stretch of atleast five (5) consecutive nucleotides selected from within theillustrative target segments are considered to be suitable for targetingas well.

Target segments can include DNA or RNA sequences that comprise at leastthe 5 consecutive nucleotides from the 5′-terminus of one of theillustrative target segments (the remaining nucleotides being aconsecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 5 to about 100 nucleotides). Similarlytarget segments are represented by DNA or RNA sequences that comprise atleast the 5 consecutive nucleotides from the 3′-terminus of one of theillustrative target segments (the remaining nucleotides being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the target segment and continuing untilthe DNA or RNA contains about 5 to about 100 nucleotides). One havingskill in the art armed with the target segments illustrated herein willbe able, without undue experimentation, to identify further targetsegments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

In embodiments of the invention the oligonucleotides bind to anantisense strand of a particular target. The oligonucleotides are atleast 5 nucleotides in length and can be synthesized so eacholigonucleotide targets overlapping sequences such that oligonucleotidesare synthesized to cover the entire length of the target polynucleotide.The targets also include coding as well as non coding regions.

In one embodiment, specific nucleic acids are targeted by antisenseoligonucleotides. Targeting an antisense compound to a particularnucleic acid, is a multistep process. The process usually begins withthe identification of a nucleic acid sequence whose function is to bemodulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a non coding polynucleotidesuch as for example, non coding RNA(ncRNA).

RNAs can be classified into (1) messenger RNAs (mRNAs), which aretranslated into proteins, and (2) non-protein-coding RNAs (ncRNAs).ncRNAs comprise microRNAs, antisense transcripts and otherTranscriptional Units (TU) containing a high density of stop codons andlacking any extensive “Open Reading Frame”. Many ncRNAs appear to startfrom initiation sites in 3′ untranslated regions (3′UTRs) ofprotein-coding loci. ncRNAs are often rare and at least half of thencRNAs that have been sequenced by the FANTOM consortium seem not to bepolyadenylated. Most researchers have for obvious reasons focused onpolyadenylated mRNAs that are processed and exported to the cytoplasm.Recently, it was shown that the set of non-polyadenylated nuclear RNAsmay be very large, and that many such transcripts arise from intergenicregions. The mechanism by which ncRNAs may regulate gene expression isby base pairing with target transcripts. The RNAs that function by basepairing can be grouped into (1) cis encoded RNAs that are encoded at thesame genetic location, but on the opposite strand to the RNAs they actupon and therefore display perfect complementarity to their target, and(2) trans-encoded RNAs that are encoded at a chromosomal locationdistinct from the RNAs they act upon and generally do not exhibitperfect base-pairing potential with their targets.

Without wishing to be bound by theory, perturbation of an antisensepolynucleotide by the antisense oligonucleotides described herein canalter the expression of the corresponding sense messenger RNAs. However,this regulation can either be discordant (antisense knockdown results inmessenger RNA elevation) or concordant (antisense knockdown results inconcomitant messenger RNA reduction). In these cases, antisenseoligonucleotides can be targeted to overlapping or non-overlapping partsof the antisense transcript resulting in its knockdown or sequestration.Coding as well as non-coding antisense can be targeted in an identicalmanner and that either category is capable of regulating thecorresponding sense transcripts—either in a concordant or disconcordantmanner. The strategies that are employed in identifying newoligonucleotides for use against a target can be based on the knockdownof antisense RNA transcripts by antisense oligonucleotides or any othermeans of modulating the desired target.

Strategy 1: In the case of discordant regulation, knocking down theantisense transcript elevates the expression of the conventional (sense)gene. Should that latter gene encode for a known or putative drugtarget, then knockdown of its antisense counterpart could conceivablymimic the action of a receptor agonist or an enzyme stimulant.

Strategy 2: In the case of concordant regulation, one couldconcomitantly knock down both antisense and sense transcripts andthereby achieve synergistic reduction of the conventional (sense) geneexpression. If, for example, an antisense oligonucleotide is used toachieve knockdown, then this strategy can be used to apply one antisenseoligonucleotide targeted to the sense transcript and another antisenseoligonucleotide to the corresponding antisense transcript, or a singleenergetically symmetric antisense oligonucleotide that simultaneouslytargets overlapping sense and antisense transcripts.

According to the present invention, antisense compounds includeantisense oligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, and otheroligomeric compounds which hybridize to at least a portion of the targetnucleic acid and modulate its function. As such, they may be DNA, RNA.DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one ormore of these. These compounds may be single-stranded, doublestranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges, mismatches or loops.Antisense compounds are routinely prepared linearly but can be joined orotherwise prepared to be circular and/or branched. Antisense compoundscan include constructs such as, for example, two strands hybridized toform a wholly or partially double-stranded compound or a single strandwith sufficient self-complementarity to allow for hybridization andformation of a fully or partially double-stranded compound. The twostrands can be linked internally leaving free 3′ or 5′ termini or can belinked to form a continuous hairpin structure or loop. The hairpinstructure may contain an overhang on either the 5′ or 3′ terminusproducing an extension of single stranded character. The double strandedcompounds optionally can include overhangs on the ends. Furthermodifications can include conjugate groups attached to one of thetermini, selected nucleotide positions, sugar positions or to one of theinternucleoside linkages. Alternatively, the two strands can be linkedvia a non-nucleic acid moiety or linker group. When formed from only onestrand, dsRNA can take the form of a self-complementary hairpin-typemolecule that doubles back on itself to form a duplex. Thus, the dsRNAscan be fully or partially double stranded. Specific modulation of gene,expression can be achieved by stable expression of dsRNA hairpins intransgenic cell lines, however, in some embodiments, the gene expressionor function is up regulated. When formed from two strands, or a singlestrand that takes the form of a self-complementary hairpin-type moleculedoubled back on itself to form a duplex, the two strands (orduplex-forming regions of a single strand) are complementary RNA strandsthat base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

In another embodiment, the desired oligonucleotides or antisensecompounds, comprise at least one of: antisense RNA, antisense DNA,chimeric antisense oligonucleotides, antisense oligonucleotidescomprising modified linkages, interference RNA (RNAi), short interferingRNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA(stRNA); or a short, hairpin RNA (shRNA); small RNA-induced geneactivation (RNAa): small activating RNAs (saRNAs), or combinationsthereof.

dsRNA can also activate gene expression, a mechanism that has beentermed “small RNA-induced gene activation” or RNAa. dsRNAs targetinggene promoters induce potent transcriptional activation of associatedgenes. RNAa was demonstrated in human cells using synthetic dsRNAs,termed “small activating RNAs” (saRNAs).

Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA)and microRNA (miRNA), have been found to be the trigger of artevolutionary conserved mechanism known as RNA interference (RNAi). RNAiinvariably leads to gene silencing. However, in instances described indetail in the examples section which follows, oligonucleotides are shownto increase the expression and/or function of the Antiviral genepolynucleotides and encoded products thereof. dsRNAs may also act assmall activating RNAs (saRNA). Without wishing to be bound by theory, bytargeting sequences in gene promoters, saRNAs would induce target geneexpression in a phenomenon referred to as dsRNA-induced transcriptionalactivation (RNAa).

In a further embodiment, the “target segments” identified herein may beemployed in a screen for additional compounds that modulate theexpression of an Antiviral gene polynucleotide. “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding an Antiviral gene and which comprise at least a5-nucleotide portion that is complementary to a target segment. Thescreening method comprises the steps of contacting a target segment of anucleic acid molecule encoding sense or natural antisensepolynucleotides of an Antiviral gene with one or more candidatemodulators, and selecting for one or more candidate modulators whichdecrease or increase the expression of a nucleic acid molecule encodingan Antiviral gene polynucleotide, e.g. SEQ ID NOS: 10 to 30. Once it isshown that the candidate modulator or modulators are capable ofmodulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding an Antiviral gene polynucleotide, themodulator may then be employed in further investigative studies of thefunction of an Antiviral gene polynucleotide, or for use as a research,diagnostic, or therapeutic agent in accordance with the presentinvention.

Targeting the natural antisense sequence modulates the function of thetarget gene. For example, the Antiviral gene (e.g. accession numbersNM_014314, NM_022168, NM_024013). In a embodiment, the target is anantisense polynucleotide of the Antiviral gene. In a embodiment, anantisense oligonucleotide targets sense and/or natural antisensesequences of an Antiviral gene polynucleotide (e.g. accession numbersNM_014314, NM_022168, NM_024013), variants, alleles, isoforms, homologs,mutants, derivatives, fragments and complementary sequences thereto.Preferably the oligonucleotide is an antisense molecule and the targetsinclude coding and noncoding regions of antisense and/or sense Antiviralgene polynucleotides.

The target segments of the present invention may be also be combinedwith their respective complementary antisense compounds of the presentinvention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications. For example, suchdouble-stranded moieties have been shown to inhibit the target by theclassical hybridization of antisense strand of the duplex to the target,thereby triggering enzymatic degradation of the target.

In a embodiment, an antisense oligonucleotide targets Antiviral genepolynucleotides (e.g. accession numbers NM_014314, NM_022168,NM_024013), variants, alleles, isoforms, homologs, mutants, derivatives,fragments and complementary sequences thereto. Preferably theoligonucleotide is an antisense molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to Antiviral gene alone but extends to any ofthe isoforms, receptors, homologs and the like of an Antiviral genemolecule.

In another embodiment, an oligonucleotide targets a natural antisensesequence of an Antiviral gene polynucleotide, for example,polynucleotides set forth as SEQ ID NO: 4 to 9, and any variants,alleles, homologs, mutants, derivatives, fragments and complementarysequences thereto. Examples of antisense oligonucleotides are set forthas SEQ ID NOS: 10 to 30.

In one embodiment, the oligonucleotides are complementary to or bind tonucleic acid sequences of an Antiviral gene antisense, including withoutlimitation noncoding sense and/or antisense sequences associated with anAntiviral gene polynucleotide and modulate expression and/or function ofan Antiviral gene molecule.

In another embodiment, the oligonucleotides are complementary to or bindto nucleic acid sequences of an Antiviral gene natural antisense, setforth as SEQ ID NO: 4 to 9 and modulate expression and/or function of anAntiviral gene molecule.

In a embodiment, oligonucleotides comprise sequences of at least 5consecutive nucleotides of SEQ ID NOS: 10 to 30 and modulate expressionand/or function of an Antiviral gene molecule.

The polynucleotide targets comprise Antiviral gene, including familymembers thereof, variants of an Antiviral gene; mutants of an Antiviralgene, including SNPs; noncoding sequences of an Antiviral gene; allelesof an Antiviral gene; species variants, fragments and the like.Preferably the oligonucleotide is an antisense molecule.

In another embodiment, the oligonucleotide targeting Antiviral genepolynucleotides, comprise: antisense RNA, interference RNA (RNAi), shortinterfering RNA (siRNA); micro interfering RNA (miRNA); a small,temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-inducedgene activation (RNAa): or, small activating RNA (saRNA).

In another embodiment, targeting of an Antiviral gene polynucleotide,e.g. SEQ ID NO: 4 to 9 modulate the expression or function of thesetargets. In one embodiment, expression or function is up-regulated ascompared to a control. In another embodiment, expression or function isdown-regulated as compared to a control.

In another embodiment, antisense compounds comprise sequences set forthas SEQ ID NOS: 10 to 30. These oligonucleotides can comprise one or moremodified nucleotides, shorter or longer fragments, modified bonds andthe like.

In another embodiment, SEQ ID NOS: 10 to 30 comprise one or more LNAnucleotides.

The modulation of a desired target nucleic acid can be carried out inseveral ways known in the art. For example, antisense oligonucleotides,siRNA etc. Enzymatic nucleic acid molecules (e.g., ribozymes) arenucleic acid molecules capable of catalyzing one or more of a variety ofreactions, including the ability to repeatedly cleave other separatenucleic acid molecules in a nucleotide base sequence-specific manner.Such enzymatic nucleic acid molecules can be used, for example, totarget virtually any RNA transcript.

Because of their sequence-specificity, trans-cleaving enzymatic nucleicacid molecules show promise as therapeutic agents for human diseaseEnzymatic nucleic acid molecules can be designed to cleave specific RNAtargets within the background of cellular RNA. Such a cleavage eventrenders the mRNA non-functional and abrogates protein expression fromthat RNA. In this manner, synthesis of a protein associated with adisease state can be selectively inhibited.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to art enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts croymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution) strategieshave been used to evolve new nucleic acid catalysts capable ofcatalyzing a variety of reactions, such as cleavage and ligation ofphosphodiester linkages and amide linkages.

The development of ribozymes that are optimal for catalytic activitywould contribute significantly to any strategy that employs RNA-cleavingribozymes for the purpose of regulating gene expression. The hammerheadribozyme, for example, functions with a catalytic rate (kcat) of about 1min−1 in the presence of saturating (10 mM) concentrations of Mg2+cofactor. An artificial “RNA ligase” ribozyme has been shown to catalyzethe corresponding self-modification reaction with a rate of about 100min−1. In addition, it is known that certain modified hammerheadribozymes that have substrate binding arms made of DNA catalyze RNAcleavage with multiple turn-over rates that approach 100 min−1. Finally,replacement of a specific residue within the catalytic core of thehammerhead with certain nucleotide analogues gives modified ribozymesthat show as much as a 10-fold improvement in catalytic rate. Thesefindings demonstrate that ribozymes can promote chemical transformationswith catalytic rates that are significantly greater than those displayedin vitro by most natural self-cleaving ribozymes. It is then possiblethat the structures of certain selfcleaving ribozymes may be optimizedto give maximal catalytic activity, or that entirely new RNA motifs canbe made that display significantly faster rates for RNA phosphodiestercleavage.

Intermolecular cleavage of an RNA substrate by an RNA catalyst that fitsthe “hammerhead” model was first shown in 1987. The RNA catalyst wasrecovered and reacted with multiple RNA molecules, demonstrating that itwas truly catalytic.

Catalytic RNAs designed based on the “hammerhead” motif have been usedto cleave specific target sequences by making appropriate base changesin the catalytic RNA to maintain necessary base pairing with the targetsequences. This has allowed use of the catalytic RNA to cleave specifictarget sequences and indicates that catalytic RNAs designed according tothe “hammerhead” model may possibly cleave specific substrate RNAs invivo.

RNA interference (RNAi) has become a powerful tool for modulating geneexpression in mammals and mammalian cells. This approach requires thedelivery of small interfering RNA (siRNA) either as RNA itself or asDNA, using an expression plasmid or virus and the coding sequence forsmall hairpin RNAs that are processed to siRNAs. This system enablesefficient transport of the pre-siRNAs to the cytoplasm where they areactive and permit the use of regulated and tissue specific promoters forgene expression.

In one embodiment, an oligonucleotide or antisense compound comprises anoligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleicacid (DNA), or a mimetic, chimera, analog or homolog thereof. This termincludes oligonucleotides composed of naturally occurring nucleotides,sugars and covalent internuclcoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftendesired over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

According to the present invention, the oligonucleotides or “antisensecompounds” include antisense oligonucleotides (e.g. RNA, DNA, mimetic,chimera, analog or homolog thereof), ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, saRNA, aRNA, andother oligomeric compounds which hybridize to at least a portion of thetarget nucleic acid and modulate its function. As such, they may be DNA,RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of oneor more of these. These compounds may be single-stranded,double-stranded, circular or hairpin oligomeric compounds and maycontain structural elements such as internal or terminal bulges,mismatches or loops. Antisense compounds are routinely prepared linearlybut can be joined or otherwise prepared to be circular and/or branched.Antisense compounds can include constructs such as, for example, twostrands hybridized to form a wholly or partially double-strandedcompound or a single strand with sufficient self-complementarity toallow for hybridization and formation of a fully or partiallydouble-stranded compound. The two strands can be linked internallyleaving tree 3′ or 5′ termini or can be linked to form a continuoushairpin structure or loop. The hairpin structure may contain an overhangon either the 5′ or 3′ terminus producing an extension of singlestranded character. The double stranded compounds optionally can includeoverhangs on the ends. Further modifications can include conjugategroups attached to one of the termini, selected nucleotide positions,sugar positions or to one of the internucleoside linkages.Alternatively, the two strands can be linked via a non-nucleic acidmoiety or linker group. When formed from only one strand, dsRNA can takethe form of a self-complementary hairpin-type molecule that doubles backon itself to form a duplex. Thus, the dsRNAs can be fully or partiallydouble stranded. Specific modulation of gene expression can be achievedby stable expression of dsRNA hairpins in transgenic cell lines. Whenformed from two strands, or a single strand that takes the form of aself-complementary hairpin-type molecule doubled back on itself to forma duplex, the two strands (or duplex-forming regions of a single strand)are complementary RNA strands that base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

The antisense compounds in accordance with this invention can comprisean antisense portion from about 5 to about 80 nucleotides (i.e. fromabout 5 to about 80 linked nucleosides) in length. This refers to thelength of the antisense strand or portion of the antisense compound, inother words, a single-stranded antisense compound of the inventioncomprises from 5 to about 80 nucleotides, and a double-strandedantisense compound of the invention (such as a dsRNA, for example)comprises a sense and an antisense strand or portion of 5 to about 80nucleotides in length. One of ordinary skill in the art will appreciatethat this comprehends antisense portions of 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides inlength, or any range therewithin.

In one embodiment, the antisense compounds of the invention haveantisense portions of 10 to 50 nucleotides in length. One havingordinary skill in the art will appreciate that this embodiesoligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length, or any range therewithin. In some embodiments,the oligonucleotides are 15 nucleotides in length.

In one embodiment, the antisense or oligonucleotide compounds of theinvention have antisense portions of 12 or 13 to 30 nucleotides inlength. One having ordinary skill in the art will appreciate that thisembodies antisense compounds having antisense portions of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length, or any range therewithin.

In another embodiment, the oligomeric compounds of the present inventionalso include variants in which a different base is present at one ormore of the nucleotide positions in the compound. For example, if thefirst nucleotide is an adenosine, variants may be produced which containthymidine, guanosine or cytidine at this position. This may be done atany of the positions of the antisense or dsRNA compounds. Thesecompounds are then tested using the methods described herein todetermine their ability to inhibit expression of a target nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 40% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identify or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

In another embodiment, the antisense oligonucleotides, such as forexample, nucleic acid molecules set forth in SEQ ID NOS: 4 to 30comprise one or more substitutions or modifications. In one embodiment,the nucleotides are substituted with locked nucleic acids (LNA).

In another embodiment, the oligonucleotides target one or more regionsof the nucleic acid molecules sense and/or antisense of coding and/ornon-coding sequences associated with Antiviral gene and the sequencesset forth as SEQ ID NOS: 1 to 9. The oligonucleotides art also targetedto overlapping regions of SEQ ID NOS: 1 to 9.

Certain oligonucleotides of this invention are chimericoligonucleotides. “Chimeric oligonucleotides” or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the target) and a region thatis a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of antisense modulation of gene expression.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligonucleotides are used, compared tophosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. In one embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in thiscase, a nucleic acid encoding ras) is routinely determined by measuringthe Tm of an oligonucleotide/target pair, which is the temperature atwhich the oligonucleotide and target dissociate; dissociation isdetected spectrophotometrically. The higher the Tm, the greater is theaffinity of the oligonucleotide for the target.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleotides and/or oligonucleotides mimetics as described above.Such; compounds have also been referred to in the art as hybrids orgapmers. Representative U.S. patents that teach the preparation of suchhybrid structures comprise, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356: and 5,700,922,each of which is herein incorporated by reference.

In another embodiment, the region of the oligonucleotide which ismodified comprises at least one nucleotide modified at the 2′ positionof the sugar, most preferably a 2′-Oalkyl, 2′O-alkyl-O-alkyl or2′-fluoro-modified nucleotide. In other embodiments, RNA modificationinclude 2′-fluoro, 2′-amino and 2′O-methyl modifications on the riboseof pyrimidines, abasic residues or an inverted base at the 3′ end of theRNA. Such modifications are routinely incorporated into oligonucleotidesand these oligonucleotides have been shown to have a higher Tm (i.e.,higher target binding affinity) than; 2′-deoxyoligonucleotides against agiven target. The effect of such increased affinity is to greatlyenhance RNAi oligonucleotide inhibition of gene expression. RNAse H is acellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes;activation of this enzyme therefore results in cleavage of the RNAtarget, and thus can greatly enhance the efficiency of RNAi inhibition.Cleavage of the RNA target can be routinely demonstrated by gelelectrophoresis. In another embodiment, the chimeric oligonucleotide isalso modified to enhance nuclease resistance. Cells contain a variety ofexo- and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance. Some desirablemodifications can be found in De Mesmacker et al. (1995) Acc. Chem.Res., 28:366-374.

Specific examples of some oligonucleotides envisioned for this inventioninclude those comprising modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH2-NH—O—CH2, CH,—N(CH3)—O—CH2 [known as amethylene(methylimino) or MMI backbone]. CH2-O—N (CH3)-CH2, CH2-N(CH3)-N (CH3)-CH2 and O—N (CH3)-CH2-CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH). The amide backbonesdisclosed by De Mesmacker et al. (1995) Acc. Chem. Res. 28:366-374 arealso preferred. Also are oligonucleotides having morpholino backbonestructures (Summerton and Weller, U.S. Pat. No. 5,034,506). In otherembodiments, such as the peptide nucleic acid (PNA) backbone, thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone. Oligonucleotidesmay also comprise one or more substituted sugar moieties,oligonucleotides comprise one of the following at the 2′ position: OH,SH, SCH3, F, OCN, OCH3 OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2 or O(CH2)nCH3 where n is from 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy,substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O—,S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3;NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino; substituted silyl; an RNA cleaving group; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide and other substituentshaving similar properties. A modification includes 2′-methoxyethoxy[2′-O—CH2 CH2 OCH3, also known as 2′-O-(2-methoxyethyl)]. Othermodifications include 2′-methoxy (2′-O—CH3), 2′-propoxy (2-OCH2 CH2CH3)and 2′-fluoro (2′-F). Similar modifications may also be made at otherpositions on the oligonucleotide, particularly the 3′ position of thesugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminalnucleotide. Oligonucleotides may also have sugar mimetics such ascyclobutyls in place of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively,nucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleotidesinclude adenine (A), guanine (G), thymine (T), cytosine (c) and uracil(U). Modified nucleotides include nucleotides found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleotides, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and2,6-diaminopurine. A “universal” base known in the art, e.g., inosine,may be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke,S. T. and Lebleu, B. eds. Antisense Research and Applications, CRCPress, Boca Raton, 1993, pp. 276-278) and are presently basesubstitutions.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, a cholesteryl moiety, athioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero- 3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid. Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides are known in the art, for example, U.S. Pat. Nos.5,138,045, 5,218,105 and 5,459,255.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes oligonucleotides which are chimericoligonucleotides as hereinbefore defined.

In another embodiment, the nucleic acid molecule of the presentinvention is conjugated with another moiety including but not limited toabasic nucleotides, polyether, polyamine, polyamides, peptides,carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in theart will recognize that these molecules can be linked to one or more ofany nucleotides comprising the nucleic acid molecule at severalpositions on the sugar, base or phosphate group.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of one of ordinary skill in the art. It is alsowell known to use similar techniques to prepare other oligonucleotidessuch as the phosphorothioates and alkylated derivatives. It is also wellknown to use similar techniques and commercially available modifiedamidites and controlled-pore glass (CPG) products such as biotin,fluorescein, acridine or psoralen-modified amidites and/or CPG(available from Glen Research, Sterling Va.) to synthesize fluorescentlylabeled, biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

In accordance with the invention, use of modifications such as the useof LNA monomers to enhance the potency, specificity and duration ofaction and broaden the routes of administration of oligonucleotidescomprised of current chemistries such as MOE, ANA, FANA, PS etc. Thiscan be achieved by substituting some of the monomers in the currentoligonucleotides by LNA monomers. The LNA modified oligonucleotide mayhave a size similar to the parent compound or may be larger orpreferably smaller. It is that such LNA-modified oligonucleotidescontain less than about 70%, more preferably less than about 60%, mostpreferably less than about 50% LNA monomers and that their sizes arebetween about 5 and 25 nucleotides, more preferably between about 12 and20 nucleotides.

Modified oligonucleotide backbones comprise, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphorates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoraroidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus containing linkages comprise, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenchydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides comprise, but are not limited to. U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds comprise, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen, et al. (1991) Science 254, 1497-1500.

In another embodiment of the invention the oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH2-NH—O—CH2-,—CH2-N (CH3)—O—CH2-known asa methylene (methylimino) or MMI backbone, —CH2O—N(CH3)-CH2-,—CH2N(CH3)-N(CH3) CH2-and-O—N(CH3)-CH2-CH2- wherein thenative phosphodiester backbone is represented as —O—P—O—CH2- of theabove referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove referenced U.S. Pat. No. 5,602,240. Also are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties, oligonucleotides comprise one of the following at the 2′position: OH; F; O—, S—, or N-alkyl; O—, S— or N-alkenyl; O—, S— orN-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C to CO alkyl or C2 to CO alkenyland alkynyl. Particularly are O (CH2)n OmCH3, O(CH2)n, OCH3, O(CH2)nNH2,O(CH2)nCH3, O(CH2)nONH2, and O(CH2nON(CH2)nCH3)2 where n and m can befrom 1 to about 10. Other oligonucleotides comprise one of the followingat the 2′ position: C to CO, (lower alkyl, substituted lower alkyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN,CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Amodification comprises 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as2′-O—(2-methoxyethyl) or 2-MOE) i.e., an alkoxyalkoxy group. A furthermodification comprises 2′-dimethylaminooxyethoxy, i.e., aO(CH2)2ON(CH3)2 group, also known as 2-DMAOE, as described in examplesherein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2-O—CH2-N(CH2)2.

Other modifications comprise 2-40 -methoxy (2′-O CH3), 2′-aminopropoxy(2′-O CH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures comprise, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and5,700,920, each of which is herein incorporated by reference.

Oligonucleotides may also comprise nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleotides comprise the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleotides comprise other synthetic andnatural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytokines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleotides comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by English et al., ‘AngewandleChemic. International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi. Y. S., Chapter 15, ‘Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B, ea. CRC Press, 1993. Certainof these nucleotides are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These comprise5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently base substitutions, even more particularly when combined with2′-Omethoxyethyl sugar modifications.

Representative United States patents that teach the preparation of theabove noted modified nucleotides as well as other modified nucleotidescomprise, but are not limited to, U.S. Pat. No. 3,687,808, as well asU.S. Pat Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177, 5,525,711;5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941,each of which is herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates, which enhance the activity, cellular distribution, orcellular uptake of the oligonucleotide.

Such moieties comprise but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates comprise, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779, 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830,5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Drug discovery: The compounds of the present invention can also beapplied in the areas of drug discovery and target validation. Thepresent invention comprehends the use of the compounds and targetsegments identified herein in drug discovery efforts to elucidaterelationships that exist between an Antiviral gene polynucleotide and adisease state, phenotype, or condition. These methods include detectingor modulating an Antiviral gene polynucleotide comprising contacting asample, tissue, cell, or organism with the compounds of the presentinvention, measuring the nucleic acid or protein level of an Antiviralgene polynucleotide and or a related phenotyptc or chemical endpoint atsome time after treatment, and optionally comparing the measured valueto a non-treated sample or sample treated with a further compound of theinvention. These methods can also be performed in parallel or incombination with other experiments to determine the function of unknowngenes for the process of target validation or to determine the validityof a particular gene product as a target for treatment or prevention ofa particular disease, condition, or phenotype.

Assessing Up-Regulation or Inhibition of Gene Expression:

Transfer of an exogenous nucleic acid into a host cell or organism canbe assessed by directly detecting the presence of the nucleic acid inthe cell or organism. Such detection can be achieved by several methodswell known in the art. For example, the presence of the exogenousnucleic acid can be detected by Southern blot or by a polymerase chainreaction (PCR) technique using primers that specifically amplifynucleotide sequences associated with the nucleic acid. Expression of theexogenous nucleic acids can also be measured using conventional methodsincluding gene expression analysis. For instance, mRNA produced from artexogenous nucleic acid can be detected and quantified using a Northernblot and reverse transcription PCR (RT-PCR).

Expression of RNA from the exogenous nucleic acid can also be detectedby measuring an enzymatic activity or a reporter protein activity. Forexample, antisense modulatory activity can be measured indirectly as adecrease or increase in target nucleic add expression as an indicationthat the exogenous nucleic acid is producing the effector RNA. Based onsequence conservation, primers can be designed and used to amplifycoding regions of the target genes. Initially, the most highly expressedcoding region from each gene can be used to build a model control gene,although any coding or non coding region can be used. Each control geneis assembled by inserting each coding region between a reporter codingregion and its poly(A) signal. These plasmids would produce an mRNA witha reporter gene in the upstream portion of the gene and a potential RNAitarget in the 3′ non-coding region. The effectiveness of individualantisense oligonucleotides would be assayed by modulation of thereporter gene. Reporter genes useful in the methods of the presentinvention include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucuronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP),cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracycline. Methods to determine modulation of areporter gene are well known in the art, and include, but are notlimited to, fluorometric methods (e.g. fluorescence spectroscopy.Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy),antibiotic resistance determination.

RIG1, MDA5, IFNA1 proteins and mRNA expression can be assayed usingmethods known to those of skill in the art and described elsewhereherein. For example, immunoassays such as the ELISA can be used tomeasure protein levels. Antiviral gene antibodies for ELISAs areavailable commercially, e.g., from R&D Systems (Minneapolis, Minn.),Abeam, Cambridge, Mass.

In embodiments, RIG1, MDA5, IFNA1 expression (e.g., mRNA or protein) ina sample (e.g., cells or tissues in vivo or in vitro) treated using anantisense oligonucleotide of the invention is evaluated by comparisonwith Antiviral gene expression in a control sample. For example,expression of the protein or nucleic acid can be compared using methodsknown to those of skill in the art with that in a mock-treated oruntreated sample. Alternatively, comparison with a sample treated with acontrol antisense oligonucleotide (e.g., one having an altered ordifferent sequence) can be made depending on the information desired. Inanother embodiment, a difference in the expression of the Antiviral geneprotein or nucleic acid in a treated vs. an untreated sample can becompared with the difference in expression of a different nucleic acid(including any standard deemed appropriate by the researcher, e.g., ahousekeeping gene) in a treated sample vs. an untreated sample.

Observed differences can be expressed as desired, e.g., in the form of aratio or fraction, for use in a comparison with control. In embodiments,the level of an Antiviral gene mRNA or protein, in a sample treated withan antisense oligonucleotide of the present invention, is increased ordecreased by about 1.25-fold to about 10-fold or more relative to anuntreated sample or a sample treated with a control nucleic acid. Inembodiments, the level of an Antiviral gene rnRNA or protein isincreased or decreased by at least about 1.25-fold, at least about1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at leastabout 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, atleast about 2-fold, at least about 2.5-fold, at least about 3-fold, atleast about 3.5-fold, at least about 1-fold, at least about 4.5-fold, atleast about 5-fold, at least about 5.5-fold, at least about 6-fold, atleast about 6.5-fold, at least about 7-fold, at least about 7.5-fold, atleast about 8-fold, at least about 8.5-fold, at least about 9-fold, atleast about 9.5-fold, or at least about 10-fold or more.

Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics, and prophylaxis, and as research reagents and componentsof kits. Furthermore, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes orto distinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics and in various biological systems, thecompounds of the present invention, either alone, or in combination withother compounds or therapeutics, are useful as tools in differentialand/or combinatorial analyses to elucidate expression patterns of aportion or the entire complement of genes expressed within cells andtissues.

As used herein the term “biological system” or “system” is defined asany organism, cell, cell culture or tissue that expresses, or is madecompetent to express products of the Antiviral genes. These include, butare not limited to, humans, transgenic animals, cells, cell cultures,tissues, xenografts, transplants and combinations thereof.

As one non limiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays. (Brazma and Vilo, (2000) FEBS Lett., 480,17-24; Celis, et al., (2000) FEBS Lett., 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., (2000) Drug Discov. Today,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, (1999) Methods Enzymol., 303, 258-72). TOGA(total gene expression analysis) (Suteliffe, et al., (2000) Proc. Natl.Acad. Sci. U.S.A., 97, 1976-81), protein arrays and proteomics (Celis,et al., (2000) FEBS Lett., 480, 2-16; Jungblut et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al.,(2000) Anal. Biochem. 286, 91-98; Larson, et al., (2000) Cytometry 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, (2000) Curr. Opin. Microbiol. 3, 316-21), comparative genomichybridization (Carulli, et al., (1998) J. Cell Biochem. Suppl., 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, (1999) Eur. J. Cancer, 35, 1895-904) and mass spectrometrymethods (To. Comb. (2000) Chem. High Throughput Screen, 3, 235-41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding an Antiviralgene. For example, oligonucleotides that hybridize with such efficiencyand under such conditions as disclosed herein as to be effectiveAntiviral gene modulators are effective primers or probes underconditions favoring gene amplification or detection, respectively. Theseprinters and probes are useful in methods requiring the specificdetection of nucleic acid molecules encoding an Antiviral gene and inthe amplification of said nucleic acid molecules for detection or foruse in further studies of an Antiviral gene. Hybridization of theantisense oligonucleotides, particularly the primers and probes, of theinvention with a nucleic acid encoding an Antiviral gene can be detectedby means known in the art. Such means may include conjugation of anenzyme to the oligonucleotide, radiolabeling of the oligonucleotide, orany other suitable detection means. Kits using such detection means fordetecting the level of an Antiviral gene in a sample may also beprepared.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs have beensafely and effectively administered to humans and numerous clinicaltrials are presently underway. It is thus established that antisensecompounds can be useful therapeutic modalities that can be configured tobe useful in treatment regimes for the treatment of cells, tissues andanimals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofan Antiviral gene polynucleotide is treated by administering antisensecompounds in accordance with this invention. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the animal in need of treatment, a therapeutically effective amountof an Antiviral gene modulator. The Antiviral gene modulators of thepresent invention effectively modulate the activity of an Antiviral geneor modulate the expression of an Antiviral gene protein. In oneembodiment, the activity or expression of an Antiviral gene in an animalis inhibited by about 10% as compared to a control. Preferably, theactivity or expression of an Antiviral gene in an animal is inhibited byabout 30%. More preferably, the activity or expression of an Antiviralgene in an animal is inhibited by 50% or more. Thus, the oligomericcompounds modulate expression of an Antiviral gene mRNA by at least 10%,by at least 50%, by as least 25%, by at least 30%, by at least 40%, byat least 50%, by at least 60%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 98%, by at least 99%, or by 100% as compared to a control.

In one embodiment, the activity or expression of an Antiviral geneand/or in an animal is increased by about 10% as compared to a control.Preferably, the activity or expression of an Antiviral gene in an animalis increased by about 30%. More preferably, the activity or expressionof an Antiviral gene in an animal is increased by 50% or more. Thus, theoligomeric compounds modulate expression of an Antiviral gene mRNA by atleast 10%, by at least 50%, by at least 25%, by at least 30%, by atleast 40%. by at least 50%, by at least 60%, by at least 70%, by atleast 75%, by at least 80%, by at least 85%, by at least 90%, by atleast 95%, by at least 98%, by at least 99%, or by 100% as compared to acontrol.

For example, the reduction of the expression of an Antiviral gene may bemeasured in serum, blood, adipose tissue, liver or any other body fluid,tissue or organ of the animal. Preferably, the cells contained withinsaid fluids, tissues or organs being analyzed contain a nucleic acidmolecule encoding Antiviral gene peptides and/or the Antiviral geneprotein itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

Conjugates: Another modification of the oligonucleotides of theinvention involves chemically linking to the oligonucleotide one or moremoieties or conjugates that enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. These moieties or conjugatescan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typicalconjugate groups includecholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of tins invention, include groups that improve uptake,enhance resistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve uptake, distribution, metabolism or excretion of thecompounds of the present invention. Representative conjugate groups aredisclosed in International Patent Application No. PCT/US92/09196, filedOct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporatedherein by reference. Conjugate moieties include, but are not limited to,lipid moieties such as a cholesterol moiety, cholic acid, a thioether,e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963, 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Formulations: The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

Although, the antisense oligonucleotides do not need to be administeredin the context of a vector in order to modulate a target expressionand/or function, embodiments of the invention relates to expressionvector constructs for the expression of antisense oligonucleotides,comprising promoters, hybrid promoter gene sequences and possess astrong constitutive promoter activity, or a promoter activity which canbe induced in the desired case.

In an embodiment, invention practice involves administering at least oneof the foregoing antisense oligonucleotides with a suitable nucleic aciddelivery system. In one embodiment, that system includes a non-viralvector operably linked to the polynucleotide. Examples of such nonviralvectors include the oligonucleotide alone (e.g. any one or more of SEQID NOS: 10 to 30) or in combination with a suitable protein,polysaccharide or lipid formulation.

Additionally suitable nucleic acid delivery systems include viralvector, typically sequence from at least one of an adenovirus,adenovirus-associated virus (AAV), helper-dependent adenovirus,retrovirus, or hemagglutinatin virus of Japan-liposome (HVJ) complex.Preferably, the viral vector comprises a strong cukaryotic promoteroperably linked to the polynucleotide e.g., a cytomegalovirus (CMV)promoter.

Additionally vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include Moloney murine leukemia virusesand HIV-based viruses. One HIV-based viral vector comprises at least twovectors wherein the gag and pol genes are from an HIV genome and the envgene is from another virus. DNA viral vectors are preferred. Thesevectors include pox vectors such as orthopox or avipox vectors,herpesvirus vectors such as a herpes simplex I virus (HSV) vector,Adenovirus Vectors and Adeno-associated Virus Vectors).

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired bioiogical activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, examples of pharmaceutically acceptable salts andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein by reference.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer: intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

For treating tissues in the central nervous system, administration canbe made by, e.g., injection or infusion into the cerebrospinal fluid.Administration of antisense RNA into cerebrospinal fluid is described,e.g., in U.S. Pat. App. Pub. No. 2007/0117772, “Methods for slowingfamilial ALS disease progression,” incorporated herein by reference inits entirety.

When it is intended that the antisense oligonucleotide of the presentinvention be administered to cells in the central nervous system,administration can be with one or more agents capable of promotingpenetration of the subject antisense oligonucleotide across theblood-brain barrier. Injection can be made, e.g., in the entorhinalcortex or hippocampus. Delivery of neurotrophic factors byadministration of an adenovirus vector to motor neurons in muscle tissueis described in, e.g., U.S. Pat. No. 6,632,427,“Adenoviral-vector-mediated gene transfer into medullary motor neurons,”incorporated herein by reference. Delivery of vectors directly to thebrain, e.g., the striatum, the thalamus, the hippocampus, or thesubstantia nigra, is known in the art and described, e.g., in U.S. Pat.No. 6,756,523, “Adenovirus vectors for the transfer of foreign genesinto cells of the central nervous system particularly in brain,”incorporated herein by reference. Administration can be rapid as byinjection or made over a period of time as by slow infusion oradministration of slow release formulations.

The subject antisense oligonucleotides can also be linked or conjugatedwith agents that provide desirable pharmaceutical or pharmacodynamicproperties. For example, the antisense oligonucleotide can be coupled toany substance, known in the art to promote penetration or transportacross the blood-brain barrier, such as an antibody to the transferrinreceptor, and administered by intravenous injection. The antisensecompound can be linked with a viral vector, for example, that makes theantisense compound more effective and/or increases the transport of theantisense compound across the blood-brain barrier. Osmotic blood brainbarrier disruption can also be accomplished by, e.g., infusion of sugarsincluding, but not limited to, meso erythritol, xylitol, D(+) galactose,D(+) lactose, D(+) xylose, duleitol, myo-inosirol, L(−) fructose, D(−)mannitol, D(+) glucose, D(+) arabinose, D(−) arabinose, cellobiose, D(+)maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(−) ribose,adonitol, D(+) arabitol, L(−) arabitol, D(+) fucose, L(−) fucose, D(−)lyxose, L(+) lyxose, and L(−) lyxose, or amino acids including, but notlimited to, glutamine, lysine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glycine, histidine, leucine, methionine,phenylalanine, proline, serine, threonine, tyrosine, valine, andtaurine. Methods and materials for enhancing blood brain barrierpenetration are described, e.g., in U.S. Pat. No. 4,866,042, “Method forthe delivery of genetic material across the blood brain barrier.” U.S.Pat. No. 6,294,520, “Material for passage through the blood-brainbarrier,” and U.S. Pat. No. 6,936,589, “Parenteral delivery systems,”all incorporated herein by reference in their entirety.

The subject antisense compounds may be admixed, encapsulated, conjugatedor otherwise associated with other molecules, molecule structures ormixtures of compounds, for example, liposomes, receptor-targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption. For example, cationic lipids maybe included in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is LIPOFECTIN (availablefrom GIBCO-BRL. Bethesda, Md.).

Oligonucleotides with at least one 2′-O-methoxyethyl modification arebelieved to be particularly useful for oral administration.Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogeneous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in citherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes that are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids. When incorporated into liposomes, these specialized lipidsresult in liposomes with enhanced circulation lifetimes relative toliposomeslacking such specialized lipids. Examples of stericallystabilized liposomes are those in which part of the vesicle-forminglipid portion of the liposome comprises one or more glycolipids or isderivatized with one or more hydrophslic polymers, such as apolyethylene glycol (PEG) moiety. Liposomes and their uses are furtherdescribed in U.S. Pat. No. 6,287,860.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating nonsurfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein by reference.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

formulations for topical administration include those in which theoligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants, lipids and liposomesinclude neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids, fatty acids andesters, pharmaceutically acceptable salts thereof, and their uses arefurther described in U.S. Pat. No. 6,287,860.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable, oral formulations are thosein which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators, surfactants include fatty acids and/or esters or saltsthereof, bile acids and/or salts thereof, bile acids/salts and fattyacids and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein by reference. Also are combinations ofpenetration enhancers, for example, fatty acids/salts in combinationwith bile acids/salts. A particularly combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or completed to form micro ornanoparticles. Oligonucleotide complexing agents and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein by reference.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapcutic agents include but are not limited tocancer chemotherapcutic drugs such as daunorubiein, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bischloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methyleyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colehicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemeitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapcutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide)Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and cortieosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. For example, the first targetmay be a particular antisense sequence of an Antiviral gene, and thesecond target may be a region from another nucleotide sequence.Alternatively, compositions of the invention may contain two or moreantisense compounds targeted to different regions of the same Antiviralgene nucleic acid target. Numerous examples of antisense compounds areillustrated herein and others may be selected from among suitablecompounds known in the art. Two or more combined compounds may be usedtogether or sequentially.

Dosing:

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC50s found to be effective inin vitro and in vivo animal models, in general, dosage is from 0.01 μgto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

In embodiments, a patient is treated with a dosage of drug that is atleast about 1, at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, at least about 10, at least about 15, at least about 20,at least about 25, at least about 30, at least about 35, are least about40, at least about 45, at least about 50, at least about 60, at leastabout 70, at least about 80. at least about 90, or at least about 100mg/kg body weight. Certain injected dosages of antisenseoligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884.“Antisense modulation of PTPIB expression” incorporated herein byreference in its entirety.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document. Applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selectedembodiments of the invention. It will be appreciated that variations inproportions and alternatives in elements of the components shown will beapparent to those skilled in the art and are within the scope ofembodiments of the present invention.

Example 1 Design of Antisense Oligonucleotides Specific for a NucleicAcid Molecule Antisense to an Antiviral Gene and/or a Sense Strand of anAntiviral Gene Polynucleotide

As indicated above the term “oligonucleotide specific for” or“oligonucleotide targets” refers to an oligonucleotide having a sequence(i) capable of forming a stable complex with a portion of the targetedgene, or (ii) capable of forming a stable duplex with a portion of anmRNA transcript of the targeted gene.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species In the case of genes that have not been sequenced.Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays

The hybridization properties of the oligonucleotides described hereincan be determined by one or more in vitro assays as known in the art.For example, the properties of the oligonucleotides described herein canbe obtained by determination of binding strength between the targetnatural antisense and a potential drug molecules using melting curveassay.

The binding strength between the target natural antisense and apotential drug molecule (Molecule) can be estimated using any of theestablished methods of measuring the strength of intermolecularinteractions, for example, a melting curve assay.

Melting curve assay determines the temperature at which a rapidtransition from double-stranded to single-stranded conformation occursfor the natural antisense/Molecule complex. This temperature is widelyaccepted as a reliable measure of the interaction strength between thetwo molecules.

A melting curve assay can be performed using a cDNA copy of the actualnatural antisense RNA molecule or a synthetic DNA or RNA nucleotidecorresponding to the binding site of the Molecule. Multiple kitscontaining all necessary reagents to performing this assay are available(e.g. Applied Biosystems Inc. MeltDoctor kit). These kits include asuitable buffer solution containing one of the double strand DNA (dsDNA)binding dyes (such as ABI HRM dyes, SYBR Green, SYTO, etc.). Theproperties of the dsDNA dyes are such that they emit almost nofluorescence in free form, but are highly fluorescent when bound todsDNA.

To perform the assay the cDNA or a corresponding oligonucleotide aremixed with Molecule in concentrations defined by the particularmanufacturer's protocols. The mixture is heated to 95° C. to dissociateall pre-formed dsDNA complexes, then slowly cooled to room temperatureor other lower temperature defined by the kit manufacturer to allow theDNA molecules to anneal. The newly formed complexes are then slowlyheated to 95° C. with simultaneous continuous collection of data on theamount of fluorescence that is produced by the reaction. Thefluorescence intensity is inversely proportional to the amounts of dsDNApresent in the reaction. The data can be collected using a real time PGRinstrument compatible with the kit (e.g., ABI's StepOne Plus Real TimePGR System or LightTyper instrument, Roche Diagnostics, Lewes, UK).

Melting peaks are constructed by plotting the negative derivative offluorescence with respect to temperature (-d(Fluorescence)/dT) on they-axis) against temperature (x-axis) using appropriate software (forexample LightTyper (Roche) or SDS Dissociation Curve, ABI). The data isanalyzed to identify the temperature of the rapid transition from dsDNAcomplex to single strand molecules. This temperature is called Tm and isdirectly proportional to the strength of interaction between the twomolecules. Typically, Tm will exceed 40° C.

Example 2 Modulation of an Antiviral Gene Polynucleotide

Treatment of HepG2 Cells with Antisense Oligonucleotides

HepG2 cells from ATCC (cat#HB-8065) were grown in growth media (MEM/EBSS(Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV) +10% FBS(Mediatech cat#MT35-011-CV)+penicillin/streptomycin (Mediatechcat#MT30002CI)) at 37° C. and 5% CO2. One day before the experiment thecells were replated at the density of 1.5×105/ml into 6 well plates andincubated at 37° C. and 5% CO2. On the day of the experiment the mediain the 6 well plates was changed to fresh growth media. All antisenseoligonucleotides were diluted to the concentration of 20 μM. Two μl ofthis solution was incubated with 400 μl of Opti-MEM media (Gibcocat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogen cat#11668019)at room temperature for 20 min and applied to each well of the 6 wellplates with HepG2 cells. A Similar mixture including 2 μl of waterinstead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO2 the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega (cat#Z3105) or RNeasy Total RNA isolation kit from Qiagen (cat#74181)following the manufacturers instructions. 600 ng of RNA was added to thereverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cat#AB1453B) or High Capacity cDNA ReverseTranscription Kit (cat#4368813) as described in the manufacturer'sprotocol. The cDNA From this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat#4369510) and primers/probes designed by ABI (AppliedBiosystems Taqman Gene Expression Assay: Hs00204833_ml. Hs00223420_ml,Hs00855471_gl by Applied Biosystems Inc., Foster City Calif.). Thefollowing PCR cycle was used: 50° C. for 2 min, 95° C. for 10 min, 40cycles of (95° C. for 15 seconds, 60° C. for 1 min) using Mx4000 thermalcycler (Stratagene).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results

Real Time PCR results show that levels of RIG1 mRNA in HepG2 cells aresignificantly increased 48 h after treatment with siRNAs to RIG1antisense hs.601664 and AK300104 (FIG. 1).

Real time PCR results show that the levels of MDA5 mRNA in HepG2 cellsare significantly increased 48 h after treatment with two of the oligosdesigned to MDA5 antisense BU663736. (FIG. 2).

Real time PCR results show that the levels of IFNA1 mRNA in HepG2 cellsare significantly increased 48 h after treatment with one oligo designedto IFNA1 antisense DA393812 (FIG. 3).

Treatment of ZR75 Cells with Antisense Oligonucleotide:

ZR75 cells from ATCC (Cat#CRL-1500) were grown in growth media (MEM/EBSS(Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV) +10% FBS(Mediatech cat#MT35-011-CV)+penicillin/streptomycin (Mediatechcat#MT30-002-CI) at 37° C. and 5% CO₂. One day before the experiment thecells were replated at the density of 1.5×10⁵/ml into 6 well plates andincubated at 37° C. and 5% CO₂. On the day of the experiment the mediain the 6 well plates was changed to fresh growth media. All antisenseoligonucleotides were diluted to the concentration of 20 μM. Two μl ofthis solution was incubated with 400 μl of Opti-MEM media (Gibcocat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogen cat#11668019)at room temperature for 20 min and applied to each well of the 6 wellplates with ZR75 cells. Similar mixture including 2 μl of water insteadof the oligonucleotide solution was used for the mock-transfectedcontrols. After 3-18 h of incubation at 37° C and 5% CO2 the media waschanged to fresh growth media. 48 h after addition of antisenseoligonucleotides the media was removed and RNA was extracted from thecells using SV Total RNA Isolation System from Promega (cat #Z3105) orRNeasy Total RNA Isolation kit from Qiagen (cat#74181) following themanufacturers' instructions. 600 ng of RNA was added to the reversetranscription reaction performed using Verso cDNA kit from ThermoScientific (cat#AB1453B) or High Capacity cDNA Reverse Transcription Kit(cat#4368813) as described in the manufacturer's protocol. The cDNA fromthis reverse transcription reaction was used to monitor gene expressionby real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) andprimers/probes designed by ABI (Applied Biosystems Taqman GeneExpression Assay: Hs00855471_gl. The following PCR cycle was used: 50°C. for 2 min, 95° C. for 10 min, 40 cycles of (95° C. for 15 seconds,60° C. for 1 min) using StepOne Plus Real Time PCR Machine (AppliedBiosystems).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results: Real time PCR results show that the levels of IFHAI mRNA inZR75 cells are significantly increased 48 h after treatment with oneoligo designed to IFNA1 antisense DA393812 (FIG. 4).

Treatment of HUVEC Cells with Antisense Oligonucleotides

HUVEC cells from ATCC (Promo Cell cat#C-12253) were grown in EpithelialGrowth Media (Promo Cell cat #C-22010) at 37° C. and 5% CO2. One daybefore the experiment the cells were repeated using Promo Cell DetachKit (cat#C-41200) at the density of 1.5×10^5/ml into 6 well plates andincubated at 37° C. and 5% CO2. On the day of the experiment the mediain the 6 well plates was changed to fresh Epithelial Growth Media. Allantisense oligonucleotides were diluted to the concentration of 20 μM.Two μl of this solution was incubated with 400 μl of Opti-MEM media(Gibco cat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogencat#11668019) at room temperature for 20 min and applied to each well ofthe 6 well plates with HUVEC cells. Similar mixture including 2 μl ofwater instead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO2 the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega (cat#Z3105) or RNeasy Total RNA Isolation kit from Qingen (cat#74181)following the manufacturers' instructions. 600 ng of RNA was added tothe reverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cat#AB1453B) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman geneExpression Mix (cat#4369510) and primers/probes designed by ABI (AppliedBiosystems Taqman Gene Expression Assays: Hs00153340_ml andHs00855471_gl by Applied Biosystems Inc., Foster City Calif.). Thefollowing PCR cycle was used: 50° C. for 2 min, 95° C. for 10 min, 40cycles of (95° C. for 15 seconds, 60° C. for 1 min) using StepOne PlusReal Time PCR Machine (Applied Biosystems Inc.) or Mx4000 thermal cycler(Stratagene).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results: Real time PCR results show that the levels of IFNA1 mRNA inHUVEC cells are significantly increased 48 h after treatment with oneoligo designed to IFNA1 antisense DA393812 (FIG. 5).

Treatment of MCF-7 Cells with Antisense Oligonucleotides:

MCF-7 cells from ATCC (cat#HTB-22) were grown in growth media (MEM/EBSS(Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV) +10% FBS(Mediatech cat#MT35-011-CV)+penicillin/streptomycin (Mediatechcat#MT30-002-CI)) at 37° C. and 5% CO2. One day before the experimentthe cells were replated at the density of 1.5×10⁵/ml into 6 well platesand incubated at 37° C. and 5% CO₂. On the day of the experiment themedia in the 6 well plates was changed to fresh growth media. Allantisense oligonucleotides were diluted to the concentration of 20 μM,Two μl of this solution was incubated with 400 μl of Opti-MEM media(Gibco cat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogencat#11668019) at room temperature for 20 min and applied to each well ofthe 6 well plates with MCF-7 cells. Similar mixture including 2 μl ofwater instead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO2 the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega (cat#Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181)following the manufacturers' instructions. 600 ng of RNA was added tothe reverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cat#AB1453B) or High Capacity cDNA ReverseTranscription Kit (cat#4368813) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat#4369510) and primers/probes designed by ABI (AppliedBiosystems Taqman Gene Expression Assay: Hs00855471_gl. The followingPCR cycle was used: 50° C. for 2 min, 95° C. for 10 min, 40 cycles of(95° C. for 15 seconds, 60° C. for 1 min) using StepOne Plus Real TimePCR Machine (Applied Biosystems).

Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the different in 18S-normalizeddCt values between treated and mock-transfected samples.

Results: ELISA Assay results show that IFNA protein as upregulated inextracts from MCF7 cells which were treated with oligos designed toIFNA1 antisense DA39381 (FIG. 6). The following table shows theoligonucleotides utilized herein.

CURNA SEQ NUMBER ID NO: SEQUENCE CUR-0544 10 AACCAGUGUAGCUGGGAGUUACUGGCACUR-0546 11 UAAGAAUUGGGUAAGAGGAAAUGGGUU CUR-0548 12CAGGAGAUUGAGACCACGGUGAAACCC CUR-0550 13 GGUGGGUUUCUGUGCCAUUUGCUGCCACUR-0552 14 UGGUUGAACACAAACUGGCUGGGUGUU CUR-0554 15AAGAGUAUCCUGUGGAGCACAGUGCGU CUR-0556 16 UUCUUAAGACUAAAGGGAAUUAUGGUUCUR-0558 17 GGGAUGAACGGUAUGACAGUUGUGGUU CUR-0560 18GCCUGUAAUCCCAGCACUUUGGGAGGC CUR-0908 19 G*C*T*T*C*T*G*A*T*G*T*C*C*TG*A*T*T*A*C CUR-0909 20 C*A*T*A*C*A*C*T*T*A*C*C*T*T*G* G*C*T*T CUR-0910 21C*C*T*C*T*A*G*C*T*A*C*T*T*C*A* A*A*T*T*C*C CUR-0911 22A*G*T*C*T*C*A*C*C*A*C*C*C*T* C*A*G*C*C*A CUR-0912 23C*T*T*C*C*T*C*C*T*G*T*C*T*T*C* T*C*C*A*C CUR-0913 24G*A*G*C*G*T*T*G*G*T*C*T*T*G*G* T*G*T*C*A*G CUR-0914 25G*C*A*G*T*T*T*C*T*G*T*T*C*A*C* T*T*C*C*C

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may he desired and advantageous for any given orparticular application.

The Abstract of the disclosure will allow the reader to quicklyascertain the nature of the technical disclosure. It is submitted withthe understanding that it will not be used to interpret or limit thescope or meaning of the following claims.

What is claimed is:
 1. A method of upregulating a function of and/or theexpression of an Antiviral gene polynucleotide in patient cells ortissues in vivo or in vitro comprising: contacting said cells or tissueswith at least one antisense oligonucleotide of 12 to 30 nucleotides inlength that targets and specifically hybridizes to a complementary 12 to30 nucleotide region of a natural antisense polynucleotide of theAntiviral gene polynucleotide selected from a IF1H1 (MDA5) gene havingSEQ ID NO: 2 wherein said natural antisense polynucleotide comprises SEQID NOS: 7 or 8; thereby upregulating a function of and/or the expressionof the Antiviral gene polynucleotide in patient cells or tissues in vivoor in vitro.
 2. The method of claim 1, wherein a function of and/or theexpression of the Antiviral gene is increased in vivo or in vitro withrespect to a control.
 3. The method of claim 1, wherein the at least oneantisense oligonucleotide targets a natural antisense polynucleotidehaving SEQ ID NO: 8 of an Antiviral gene polynucleotide.
 4. The methodof claim 1, wherein the at least one antisense oligonucleotide targets anatural antisense polynucleotide antisense to coding and/or non-codingnucleic acid sequences of an Antiviral gene polynucleotide.
 5. Themethod of claim 1, wherein the at least one antisense oligonucleotidetargets a natural antisense polynucleotide having overlapping and/ornon-overlapping sequences with an Antiviral gene polynucleotide.
 6. Themethod of claim 1, wherein the at least one antisense oligonucleotidecomprises one or more modifications selected from: at least one modifiedsugar moiety, at least one modified intemucleoside linkage, at least onemodified nucleotide, and combinations thereof.
 7. The method of claim 6,wherein the one or more modifications comprise at least one modifiedsugar moiety selected from: a 2′-O-methoxyethyl modified sugar moiety, a2′-methoxy modified sugar moiety, a 2′-O-alkyl modified sugar moiety, abicyclic sugar moiety, and combinations thereof.
 8. The method of claim6, wherein the one or more modifications comprise at least one modifiedintemucleoside linkage selected from: a phosphorothioate,alkylphosphonate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,carboxymethyl ester, and combinations thereof.
 9. The method of claim 6,wherein the one or more modifications comprise at least one modifiednucleotide selected from: a peptide nucleic acid (PNA), a locked nucleicacid (LNA), an arabino-nucleic acid (FANA), an analogue, a derivative,and combinations thereof.
 10. The method of claim 1, wherein the atleast one oligonucleotide comprises at least one oligonucleotidesequences set forth as SEQ ID NOS: 19 to 26.